Exosomal loading using hydrophobically modified oligonucleotides

ABSTRACT

In one aspect, the invention relates to a method of loading exosomes with oligonucleotide cargo, by incubating an oligonucleotide comprising one or more hydrophobic modifications with a population of exosomes for a period of time sufficient to allow loading of the exosomes with the oligonucleotide. Exosomes loaded with hydrophobically modified oligonucleotide cargo, and uses thereof, are also provided.

RELATED APPLICATIONS

This application is a Continuation Application of U.S. patentapplication Ser. No. 15/304,943, filed on Oct. 18, 2016; which is a 35U.S.C. § 371 national stage filing of International Patent ApplicationNo. PCT/US2015/026350, filed on Apr. 17, 2015; which claims priority toU.S. Provisional Patent Application No. 61/981,722, filed on Apr. 18,2014. The entire contents of each of the foregoing applications areincorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant numberTR000888 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 23, 2020, isnamed SL 122274_14003.txt and is 4,529 bytes in size.

BACKGROUND OF THE INVENTION

Exosomes are extracellular particles produced by cells which caneffectively transfer a variety of molecules, including small ribonucleicacids (RNAs), from cell to cell. Exosomes have been used as deliveryvehicles to transfer synthetic small RNAs, e.g., small interfering RNAs(siRNAs), to cells and tissues of interest in a proficient manner.Currently, however, the commercial potential of exosomes as deliveryvehicles is minimal, due primarily to highly inefficient andnon-scalable methods used for loading synthetic nucleic acids intoexosomes.

SUMMARY OF THE INVENTION

Current methods of loading exosomes with nucleic acid cargo includeelectroporation, or transfection with cationic lipid reagents. Othermethods include loading by ultracentrifugation. Each of these methodsresults in very low loading efficiency, where only a small fraction ofadded oligonucleotide molecules are transferred to exosomes. Forexample, one laboratory reported a loading efficiency usingultracentrifugation of 1 molecule of siRNA per 1000 exosomes.

New methods of loading exosomes with nucleic acid cargo are describedherein. These methods are based, at least in part, on the discovery thatintroduction of a hydrophobic modification into an oligonucleotidefacilitates exosomal loading. Accordingly, in some embodiments, thepresent invention relates to improved methods of loading exosomes withnucleic acid cargo. In other embodiments, the invention relates toexosomes loaded with nucleic acid cargo, in which the nucleic acidmolecules contain one or more hydrophobic modifications. In otherembodiments, the invention relates to the use of exosomes loaded withhydrophobically-modified nucleic acid cargo as delivery vehicles, e.g.,for delivery of small oligonucleotides to cells or tissues.

Accordingly, in one aspect, the invention relates to a method of loadingexosomes with oligonucleotide cargo, by incubating an oligonucleotidecomprising one or more hydrophobic modifications with a population ofexosomes. The hydrophobically modified oligonucleotide and thepopulation of exosomes are incubated for a period of time sufficient toallow loading of the exosomes with the oligonucleotide. In preferredembodiments, the method takes place in the absence of electroporation,cationic liposome transfection reagents, and/or ultracentrifugation.

In another aspect, the invention provides a method of loading exosomeswith oligonucleotide cargo, consisting essentially of incubating anoligonucleotide comprising one or more hydrophobic modifications with apopulation of exosomes.

The methods described herein are highly efficient and easily scalable,and allow the rapid production of oligonucleotide-loaded exosomes inquantities needed for therapeutic administration.

In certain embodiments of the foregoing aspects, loading of the exosomeswith the oligonucleotide occurs in 30 minutes or less, e.g., 5 minutesor less. In some embodiments, loading of the exosomes occurs in 30minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute.

The methods described herein allow exosomes to be loaded witholigonucleotide cargo at efficiencies that were not achievable usingtraditional methods. Accordingly, in one embodiment, at least 80% of theexosomes incubated with an oligonucleotide comprising one or morehydrophobic modifications are loaded with the oligonucleotide. In apreferred embodiment, at least 90% of the exosomes incubated with anoligonucleotide comprising one or more hydrophobic modifications areloaded with the oligonucleotide. In exemplary embodiments, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or more of the exosomes incubated with anoligonucleotide comprising one or more hydrophobic modifications areloaded with the oligonucleotide. In one embodiment at least 99% of theexosomes incubated with an oligonucleotide comprising one or morehydrophobic modifications are loaded with the oligonucleotide.

The methods described herein also allow exosomes to be loaded withlarger quantities of oligonucleotide cargo than was achievable usingtraditional methods. Accordingly, in one embodiment, exosomes are loadedwith an average of at least 500 hydrophobically modifiedoligonucleotides per exosome, e.g., at least 600, at least 700, at least800, at least 900, at least 1000, at least 1200, at least 1500, at least2000, at least 2500, at least 3000 or more hydrophobically modifiedoligonucleotides per exosome. In one embodiment, exosomes are loadedwith an average of about 500-3000 hydrophobically modifiedoligonucleotides per exosome. In another embodiment, exosomes are loadedwith an average of about 500-1000 hydrophobically modifiedoligonucleotides per exosome. In another embodiment, exosomes are loadedwith an average of about 1000-1500 hydrophobically modifiedoligonucleotides per exosome. In another embodiment, exosomes are loadedwith an average of about 1000-3000 hydrophobically modifiedoligonucleotides per exosome. In another embodiment, exosomes are loadedwith up to about 3000 hydrophobically modified oligonucleotides perexosome.

In preferred embodiments, the oligonucleotide is a syntheticoligonucleotide. In some embodiments, the oligonucleotide is a siRNA,siRNA-GalNAc, antisense RNA, LNA, hairpin siRNA, PMO, miRNA, miRNAinhibitor, or a combination thereof. In an exemplary embodiment, theoligonucleotide is siRNA. In another embodiment, the oligonucleotide ismiRNA.

In certain embodiments of the foregoing aspects, the hydrophobicmodification increases the hydrophobicity of the oligonucleotide by atleast about 2 orders of magnitude relative to unmodifiedoligonucleotide.

In certain embodiments, the hydrophobic modification is a backbonemodification, a sugar modification (e.g., a ribose modification), a basemodification, or a combination thereof. Backbone modifications caninclude, in some embodiments, phosphorothioate modifications,phosphorodithioate modifications, p-ethoxy modifications,methylphosphonate modifications, methylphosphorothioate modifications,alkyl- and aryl-phosphate modifications, alkylphosphotriestermodifications, peptide nucleic acid (PNA) modifications, and/or lockednucleic acid (LNA) modifications. Ribose modifications can include, insome embodiments, 2′O-Methyl, 2′Methoxy-ethyl, 2′Fluor, or 2′FANA. Basemodifications can include, in some embodiments, phenyl, naphthyl, orisobutyl. In addition to increasing the hydrophobic character ofoligonucleotide cargo, the foregoing modifications increase thestability of the oligonucleotide cargo in the presence of exosomes andmake the oligonucleotide cargo resistant to degradation. In someembodiments, at least 30% of the nucleotides in the oligonucleotidecontain one or more backbone modifications, sugar modifications, and/orbase modifications. For example, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65% or more of the nucleotides in the oligonucleotide cargo can containone or more backbone modifications, sugar modifications, and/or basemodifications. In an exemplary embodiment, at least 50% of thenucleotides in the oligonucleotide contain one or more backbonemodifications, sugar modifications, and/or base modifications.

In certain embodiments, the oligonucleotide can be conjugated to one ormore hydrophobic moieties. For example, in some embodiments thehydrophobic moiety can be a sterol, GM1, a lipid, a vitamin, a smallmolecule, or a peptide, or a combination thereof. In an exemplaryembodiment, the oligonucleotide cargo is conjugated to a sterol, e.g.,cholesterol. In another exemplary embodiment, the oligonucleotide cargois conjugated to GM1. In another exemplary embodiment, theoligonucleotide cargo is conjugated to myristic acid, or a derivativethereof.

In exemplary embodiments, the oligonucleotide cargo is stabilized byincorporation of one or more backbone modifications, sugarmodifications, and/or base modifications as described herein, andadditionally is conjugated to a hydrophobic moiety. For example, in oneembodiment, the oligonucleotide cargo contains one or more backbonemodifications, sugar modifications, and/or base modifications to atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65% or more of the nucleotides, andfurther is conjugated to a hydrophobic moiety, e.g., a sterol, GM1, alipid, a vitamin, a small molecule, or a peptide, or a combinationthereof. In an exemplary embodiment, the oligonucleotide cargo isconjugated to a sterol, e.g., cholesterol. In another exemplaryembodiment, the oligonucleotide cargo is conjugated to GM1. In anotherexemplary embodiment, the oligonucleotide cargo is conjugated tomyristic acid, or a derivative thereof. In one embodiment, theoligonucleotide cargo is an siRNA that is modified as depicted in FIG.10.

In certain embodiments, the exosomes can be derived from cultured cells.In exemplary embodiments, the exosomes can be derived from dendriticcells (DC), B cells, T cells, mast cells, epithelial cells, stem cells,neuronal cells, or tumor cells, or combinations thereof. In otherembodiments, the exosomes can be derived from immature dendritic cellsor induced pluripotent stem cells (iPS cells). In other embodiments, theexosomes are derived from neuronal cells. Alternatively, in someembodiments, the exosomes are synthetic exosomes.

In some embodiments, the exosomes can contain a targeting peptide, forexample, a targeting peptide that directs the exosomes to neuronalcells.

In some embodiments, exosomes are purified after loading withhydrophobically modified oligonucleotide to separate the exosomes fromunloaded oligonucleotide. In some embodiments, exosomes can be purifiedby ultrafiltration. In other embodiments, the exosomes can be purifiedby differential centrifugation.

In certain embodiments, the oligonucleotide directs the silencing of amutant huntingtin gene or a mutant SOD1 gene. For example, in someembodiments, the oligonucleotide is an siRNA targeting a mutanthuntingtin gene or a mutant SOD1 gene.

In another aspect, the invention provides compositions comprisingexosomes loaded with hydrophobically modified oligonucleotide, wherebythe compositions are produced by way of any of the foregoing methods.For example, in one embodiment, the invention provides a compositioncomprising exosomes loaded with hydrophobically modifiedoligonucleotide, wherein the exosomes are loaded by incubating anoligonucleotide comprising one or more hydrophobic modifications with apopulation of exosomes for a period of time sufficient to allow loadingof the exosomes with the oligonucleotide.

In another aspect, the invention provides a composition comprising aplurality of exosomes loaded with an oligonucleotide comprising one ormore hydrophobic modifications.

In some embodiments, at least 80% of the exosomes are loaded with thehydrophobically modified oligonucleotide. In a preferred embodiment, atleast 90% of the exosomes are loaded with the hydrophobically modifiedoligonucleotide. In exemplary embodiments, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or more of the exosomes are loaded with the hydrophobicallymodified oligonucleotide. In one embodiment at least 99% of the exosomesare loaded with the hydrophobically modified oligonucleotide.

In other embodiments, the foregoing compositions contain exosomes thatare loaded with an average of at least 500 hydrophobically modifiedoligonucleotides per exosome, e.g., at least 600, at least 700, at least800, at least 900, at least 1000, at least 1200, at least 1500, at least2000, at least 2500, at least 3000 or more hydrophobically modifiedoligonucleotides per exosome. In one embodiment, the exosomes contain anaverage of about 500-3000 hydrophobically modified oligonucleotides perexosome. In another embodiment, exosomes contain an average of about500-1000 hydrophobically modified oligonucleotides per exosome. Inanother embodiment, exosomes contain an average of about 1000-1500hydrophobically modified oligonucleotides per exosome. In anotherembodiment, exosomes contain an average of about 1000-3000hydrophobically modified oligonucleotides per exosome. In anotherembodiment, exosomes contain up to about 3000 hydrophobically modifiedoligonucleotides per exosome.

In preferred embodiments, the oligonucleotide is a syntheticoligonucleotide. In some embodiments, the oligonucleotide is a siRNA,siRNA-GalNAc, antisense RNA, LNA, hairpin siRNA, PMO, miRNA, miRNAinhibitor, or a combination thereof. In an exemplary embodiment, theoligonucleotide is siRNA. In another embodiment, the oligonucleotide ismiRNA.

In certain embodiments of the foregoing aspects, the hydrophobicmodification increases the hydrophobicity of the oligonucleotide by atleast about 2 orders of magnitude relative to unmodifiedoligonucleotide.

In certain embodiments, the hydrophobic modification is a backbonemodification, a sugar modification (e.g., a ribose modification), a basemodification, or a combination thereof. Backbone modifications caninclude, in some embodiments, phosphorothioate modifications,phosphorodithioate modifications, p-ethoxy modifications,methylphosphonate modifications, methylphosphorothioate modifications,alkyl- and aryl-phosphate modifications, alkylphosphotriestermodifications, peptide nucleic acid (PNA) modifications, and/or lockednucleic acid (LNA) modifications. Ribose modifications can include, insome embodiments, 2′O-Methyl, 2′Methoxy-ethyl, 2′Fluor, or 2′FANA. Basemodifications can include, in some embodiments, phenyl, naphthyl, orisobutyl. In addition to increasing the hydrophobic character ofoligonucleotide cargo, the foregoing modifications increase thestability of the oligonucleotide cargo in the presence of exosomes andmake the oligonucleotide cargo resistant to degradation. In someembodiments, at least 30% of the nucleotides in the oligonucleotidecontain one or more backbone modifications, sugar modifications, and/orbase modifications. For example, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65% or more of the nucleotides in the oligonucleotide cargo can containone or more backbone modifications, sugar modifications, and/or basemodifications. In an exemplary embodiment, at least 50% of thenucleotides in the oligonucleotide contain one or more backbonemodifications, sugar modifications, and/or base modifications.

In certain embodiments, the oligonucleotide can be conjugated to one ormore hydrophobic moieties. For example, in some embodiments thehydrophobic moiety can be a sterol, GM1, a lipid, a vitamin, a smallmolecule, or a peptide, or a combination thereof. In an exemplaryembodiment, the oligonucleotide cargo is conjugated to a sterol, e.g.,cholesterol. In another exemplary embodiment, the oligonucleotide cargois conjugated to GM1. In another exemplary embodiment, theoligonucleotide cargo is conjugated to myristic acid, or a derivativethereof.

In exemplary embodiments, the oligonucleotide cargo is stabilized byincorporation of one or more backbone modifications, sugarmodifications, and/or base modifications as described herein, andadditionally is conjugated to a hydrophobic moiety. For example, in oneembodiment, the oligonucleotide cargo contains one or more backbonemodifications, sugar modifications, and/or base modifications to atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65% or more of the nucleotides, andfurther is conjugated to a hydrophobic moiety, e.g., a sterol, GM1, alipid, a vitamin, a small molecule, or a peptide, or a combinationthereof. In an exemplary embodiment, the oligonucleotide cargo isconjugated to a sterol, e.g., cholesterol. In another exemplaryembodiment, the oligonucleotide cargo is conjugated to GM1. In anotherexemplary embodiment, the oligonucleotide cargo is conjugated tomyristic acid, or a derivative thereof. In one embodiment, theoligonucleotide cargo is an siRNA that is modified as depicted in FIG.10.

In certain embodiments, the exosomes can be derived from cultured cells.In exemplary embodiments, the exosomes can be derived from dendriticcells (DC), B cells, T cells, mast cells, epithelial cells, stem cells,neuronal cells, or tumor cells, or combinations thereof. In otherembodiments, the exosomes can be derived from immature dendritic cellsor induced pluripotent stem cells (iPS cells). In other embodiments, theexosomes are derived from neuronal cells. Alternatively, in someembodiments, the exosomes are synthetic exosomes.

In some embodiments, the exosomes can contain a targeting peptide, forexample, a targeting peptide that directs the exosomes to neuronalcells.

In some embodiments, the oligonucleotide targets a gene associated witha neurological disease or disorder. In certain embodiments, theoligonucleotide directs the silencing of a mutant huntingtin gene or amutant SOD1 gene. For example, in some embodiments, the oligonucleotideis an siRNA targeting a mutant huntingtin gene or a mutant SOD1 gene.

In some embodiments, the foregoing compositions can be produced byincubating an oligonucleotide comprising one or more hydrophobicmodifications with a population of exosomes.

In another aspect, the invention provides a pharmaceutical compositionfor delivery of a therapeutic oligonucleotide to a subject, comprisingexosomes loaded with a therapeutically effective amount of anoligonucleotide containing one or more hydrophobic modifications, and apharmaceutically acceptable carrier or excipient.

In another aspect, the invention provides a pharmaceutical compositionthat comprises exosomes loaded with a therapeutically effective amountof an oligonucleotide comprising one or more hydrophobic modifications,and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the pharmaceutical compositions contain at leastabout 10⁷ exosomes, e.g., at least about 10⁸ exosomes, 10⁹ exosomes,10¹⁰ exosomes, 10¹¹ exosomes, 10¹² exosomes, 10¹³ exosomes, 10¹⁴exosomes, 10¹⁵ exosomes, 10¹⁶ exosomes, 10¹⁷ exosomes, 10¹⁸ exosomes, or10¹⁹ exosomes. In an exemplary embodiments, the pharmaceuticalcompositions contain about 10⁸-10¹⁵ exosomes.

In some embodiments, at least about 90% of the exosomes in thecomposition are loaded with hydrophobically modified oligonucleotide. Inother embodiments, at least about 99% of the exosomes in the compositionare loaded with hydrophobically modified oligonucleotide. The exosomesin the pharmaceutical compositions can be loaded with thehydrophobically modified oligonucleotide at concentrations describedherein. For example, the exosomes may contain an average of about500-1000 oligonucleotide molecules per exosome. In other embodiments,the exosomes may contain an average of about 1000-3000 oligonucleotidemolecules per exosome.

In another embodiment, the invention provides a pharmaceuticalcomposition containing a plurality of exosomes loaded with ahydrophobically modified oligonucleotide, as set forth in anyembodiments described herein, and a pharmaceutically acceptable carrieror excipient.

In another aspect, the invention provides a method of delivering anexogenous oligonucleotide to a subject, by administering to the subjecta composition comprising exosomes, wherein the exosomes are loaded withan exogenous oligonucleotide containing one or more hydrophobicmodifications.

In another aspect, the invention provides a method of silencing geneexpression in a cell, by contacting the cell with a compositioncomprising exosomes, wherein the exosomes are loaded with anoligonucleotide that directs the silencing of a target gene in the cell,and wherein the oligonucleotide contains one or more hydrophobicmodifications. In certain embodiments, the target gene is associatedwith a neuronal disease or disorder. In exemplary embodiments, thetarget gene is a mutant huntingtin gene or a mutant SOD1 gene.

In another aspect, the invention provides a method of silencing geneexpression in a subject, by administering to the subject a compositioncomprising exosomes, wherein the exosomes are loaded with anoligonucleotide that directs the silencing of a target gene in thesubject, and wherein the oligonucleotide contains one or morehydrophobic modifications. In certain embodiments, the target gene isassociated with a neuronal disease or disorder. In exemplaryembodiments, the target gene is a mutant huntingtin gene or a mutantSOD1 gene.

In another aspect, the invention provides a method of treating a diseaseor disorder in a subject, by administering to the subject a compositioncontaining exosomes loaded with hydrophobically modified oligonucleotidecargo, wherein the hydrophobically modified oligonucleotide cargoreduces or inhibits expression of a gene associated with a disease ordisorder. In some embodiments, the disorder is a neurological disorder.In some embodiments, the disorder is Huntington's disease, and theoligonucleotide cargo reduces or inhibits expression of a mutanthuntingtin gene. In other embodiments, the disorder is ALS, and theoligonucleotide cargo reduces or inhibits expression of a mutant SOD1gene.

The foregoing methods can employ exosomes loaded with hydrophobicallymodified oligonucleotides as described herein.

For example, in one embodiment of the foregoing aspects, at least 90% ofexosomes in the composition are loaded with the oligonucleotide. Inother embodiments, at least 99% of the exosomes are loaded with theoligonucleotide. In one embodiment, the exosomes are loaded with theoligonucleotide at an average concentration of about 500-1000oligonucleotide molecules per exosome. In another embodiment, theexosomes are loaded with the oligonucleotide at an average concentrationof about 1000-3000 oligonucleotide molecules per exosome.

In preferred embodiments of the foregoing aspects, the oligonucleotideis a synthetic oligonucleotide. In some embodiments, the oligonucleotideis a siRNA, siRNA-GalNAc, antisense RNA, LNA, hairpin siRNA, PMO, miRNA,miRNA inhibitor, or a combination thereof. In an exemplary embodiment,the oligonucleotide is siRNA. In another embodiment, the oligonucleotideis miRNA.

In certain embodiments of the foregoing aspects, the hydrophobicmodification increases the hydrophobicity of the oligonucleotide by atleast about 2 orders of magnitude relative to unmodifiedoligonucleotide. In some embodiments, at least 30% of the nucleotides inthe oligonucleotide contain a hydrophobic modification. In otherembodiments, at least 50% of the nucleotides in the oligonucleotidecontain a hydrophobic modification.

In certain embodiments, the hydrophobic modification is a backbonemodification, a sugar modification (e.g., a ribose modification), a basemodification, or a combination thereof. Backbone modifications caninclude, in some embodiments, phosphorothioate modifications,phosphorodithioate modifications, p-ethoxy modifications,methylphosphonate modifications, methylphosphorothioate modifications,alkyl- and aryl-phosphate modifications, alkylphosphotriestermodifications, peptide nucleic acid (PNA) modifications, and/or lockednucleic acid (LNA) modifications. Ribose modifications can include, insome embodiments, 2′O-Methyl, 2′Methoxy-ethyl, 2′Fluor, or 2′FANA. Basemodifications can include, in some embodiments, phenyl, naphthyl, orisobutyl.

In certain embodiments, the oligonucleotide can be conjugated to one ormore hydrophobic moieties. For example, in some embodiments thehydrophobic moiety can be a sterol, GM1, a lipid, a vitamin, a smallmolecule, or a peptide, or a combination thereof. In an exemplaryembodiment, the oligonucleotide cargo is conjugated to a sterol, e.g.,cholesterol. In another exemplary embodiment, the oligonucleotide cargois conjugated to GM1. In another exemplary embodiment, theoligonucleotide cargo is conjugated to myristic acid, or a derivativethereof. In one embodiment, the oligonucleotide cargo is an siRNA thatis modified as depicted in FIG. 10.

In certain embodiments, the exosomes can be derived from cultured cells.In exemplary embodiments, the exosomes can be derived from dendriticcells (DC), B cells, T cells, mast cells, epithelial cells, stem cells,neuronal cells, or tumor cells, or combinations thereof. In otherembodiments, the exosomes can be derived from immature dendritic cellsor induced pluripotent stem cells (iPS cells). In other embodiments, theexosomes are derived from neuronal cells. Alternatively, in someembodiments, the exosomes are synthetic exosomes.

In some embodiments, the exosomes can contain a targeting peptide, forexample, a targeting peptide that directs the exosomes to neuronalcells.

In another embodiment of the foregoing aspects, the exosomes loaded withhydrophobically modified oligonucleotide cargo are produced by a methodcomprising incubating the hydrophobically modified oligonucleotide witha population of exosomes.

In various embodiments of any of the foregoing aspects, thehydrophobically modified oligonucleotide can contain a detectable label.Exemplary labels include fluorescent labels and/or radioactive labels.In one embodiment, the detectable label is Cy3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B. Purification and QC of exosomes produced from U87conditioned media. A. Overview of differential centrifugation protocol.B. Particle size and concentration evaluation by Nanoparticle trackinganalyzer (Nanosight) C. Electron microscopy of exosome sample confirmingsize and integrity.

FIG. 2A-D. Efficient loading of hydrophobically modified siRNA (hsiRNA)into Exosomes. A. Loading and purification scheme. B. % of DY547-hsiRNAbound to exosomes and free in solution after ultracentrifugation weremeasured by nandrop/Q-RT-PCR. ˜25% of hsiRNA were bound to exosomesstably as less than 5% is released after the second spin. No hsiRNA wereprecipitated in the absence of exosomes. C. Nanosight profile ofexosomes before and after hsiRNA loading (treated in parallel). Inpresence of exosomes we observe a clear increase in size and shift inthe zeta potential (Melvern). D. Electron microscopy of Exosomes loadedwith Biotinylated Htt-hsiRNA detected by streptavidin/Immunogoldparticles used to detect the biotin-hsiRNA. The arrows indicate exosomeswith biotin-hsiRNA on surface.

FIG. 3A-B. Examples of hydrophobically modified oligonucleotides. Anexample of hydrophobically modified asymmetric siRNAs, siRNA-sterolconjugates, siRNA-GalNac conjugates, 2′F, 2′ O-methyl modified siRNAs,phosphorothioated ASOs, LNAs, methoxyethyl, phenol, isobutyl, andnaphthyl modifications.

FIG. 4A-C. Optimization of exosome loading with RNAi agents usingSephacryl S-1000 Fractionation. Exosomes purified from U87 cells andpurified by ultracentrifugation were loaded with in the presence of 10μM hsiRNA (NTC and htt targeting). Samples were fractionated in PBS onSephacryl S-1000 at 0.5 ml/min A Profile of absorbance at 260 nm ofnon-loaded exosomes. B. Profile of absorbance at 260 nm of exosomesloaded with hsiRNA NTC.C. Profile of absorbance at 260 nm of exosomesloaded with hsiRNA htt. Efficient loading (26-42%) was observed.

FIG. 5A-D. hsiRNA-Exosome Complexes: Passive vs. Exosome-mediated hsiRNAUptake in HeLa cells. U87 cell-derived exosomes were purified bydifferential centrifugation and labeled by PKH67 dye (Sigma). HeLa cellswere treated with exosomes (labeled with PKH67), hsiRNA and hsiRNApre-formulated with exosomes. A. Exosomes alone (PKH67), 24 hours. B.hsiRNA alone, 12 hours; membrane and cytoplasmic staining C.hsiRNA-loaded exosomes, 12 hours; clear asymmetric peri-nuclearstaining. D. hsiRNA (Cy3)-loaded exosomes (PKH67); degree ofco-localization. Exosomes=green (PKH67 dye); hsiRNA=red (Cy3);nucleus=blue (dapi)

FIGS. 6A and B. Comparison of silencing efficiency: hsiRNA passive vs.exosome-mediated hsiRNA functionality test in HeLa cells. U87cell-derived exosomes were purified by differential centrifugation. A.HeLa were treated with hsiRNA-PPIB alone or PPIB hsiRNA into loadedexosomes. The level of PPIB silencing was measured at 72 hours, usingHIT as housekeeping (QuantiGene 2.0 assay, Affimetrix). B. Comparison ofsilencing efficiency of PPIB hsiRNA alone or mediated by exosomes.

FIGS. 7A and B. hsiRNA-loaded exosomes in primary neurons: uptake andfunctionality monitoring of hsiRNA targeting PPIB and HTT mRNA. A.Exosomes from U87 cells were labeled with PKH67 (Sigma), loaded withCy3-labeled hsiRNA (PPIB) and added at 10⁸ per well. Each well contained˜2*10⁶ cell per well. Exosomes=green (PKH67 dye); hsiRNA=red (Cy3);nucleus=blue (dapi). B. Exosomes purified from U87 cells by standarddifferential centrifugation were loaded with hsiRNA against huntingtinmRNA: incubation for 1 hr at 37° C. and ultra-centrifuged. Loadedexosomes were then transferred onto WT (FVB) Primary Cortical Neurons(1×10⁵/well), followed by a week of incubation. Level of silencing wasdetermined by QuantiGene Assay (housekeeping gene=PPIB; n=3, error is aSTDEV).

FIGS. 8A and B. In vivo distribution of hsiRNA-loaded exosomes in mousebrain. Exosomes purified from U87 cells were loaded with Cy3-labeledhsiRNA against HTT mRNA. A single injection of exosome-hsiRNA complexeswas performed on mouse brain. Mice were sacrificed 24 h or 72 h afterthe injection. Neurons=NeuN staining (neuronal marker; green);hsiRNA=red (Cy3). A. Visualization of injected side (left) andnon-injected side (right) of mouse brain by fluorescence microscopy. B.Visualization of brain sections from injected side (upper panel) andnon-injected side (lower panel) of mouse brain fixed after 72 h afterexosome-hsiRNA complex injection by fluorescence confocal microscopy.

FIG. 9A-D. Exosome purification and characterization. (A) Schematic foran exosome purification procedure based on differentialultracentrifugation (Thery et al, 2006.). The speed and length of eachcentrifugation are indicated to the right of the arrows. After the firsttwo centrifugations, pellets (cells, dead cells and cell debris) arediscarded, and the supernatant is kept for the next step. In contrast,after the two 100,000 g centrifugations, pellets (exosomes+contaminantproteins, exosomes) are kept, and supernatants are discarded. (B)Quantification and size monitoring of U87 MG-derived vesicles byNanoparticle Tracking Analysis. Particle concentration is shown as afunction of particle size. (C) Electron microscopy validates vesicleintegrity. (D) U87 glioblastoma cells were cultured in exosome-freemedium for 3 days. Exosomes were purified from conditioned medium asdescribed above. Following purification, 50 μl of conditioned medium orexosome sample were analyzed by size-exclusion chromatography.

FIG. 10A-C. Efficient loading of exosomes with hydrophobically modifiedsiRNAs (hsiRNAs). (A) The hsiRNAs used in this experiment are asymmetriccompounds, with a short duplex region (15 base-pairs) andsingle-stranded fully phosphorothioated tail, where all pyrimidines aremodified with 2′-fluoro and 2′-O-methyl modifications and the 3′ end ofthe passenger strand is conjugated to Teg-Cholesterol. (B) Co-incubationof hsiRNAs and exosomes (U87-derived) results in ˜1000 to 3000 hsiRNAassociation per exosome. hsiRNA-loaded exosomes were separated byultracentrifugation. The pellet (pink) contains Cy3-hsiRNA-loadedexosomes. (C) Percent of hsiRNAs in the pellet (black bar) vs in thesolution (grey bar) after ultracentrifugation of hsiRNAs in the presenceand absence of exosomes indicates that ˜30% of hsiRNAs are associatedwith exosomes only in the presence of exosomes (n=3, standard deviationshown).

FIG. 11A-C. Cholesterol promotes efficient loading and interaction ofhsiRNA into exosomes. (A) Cy3-HTT10150-loaded exosome fluorescence wasmeasured by spectrophotometer. After ultracentrifugation 1,approximately 20% of hsiRNA are associated with the exosomes (black),while non formulated hsiRNA do not pellet. After ultraspin 2, majorityof hsiRNA remains associated with the exosomes (n=3 replicates;mean±S.D.). (B) Representative pictures of ultracentrifugednon-formulated hsiRNA (left tube) or hsiRNA-loaded exosomes (righttube). (C) Representative pictures of ultracentrifuged siRNA-loadedexosomes (left tube) or corresponding hsiRNA-loaded exosomes (righttube).

FIG. 12A-C. Characterization of unloaded and hsiRNA-loaded exosomes.Exosomes were loaded with hsiRNAs and purified by ultracentrifugation.Loaded and unloaded exosomes were characterized for (A) charge(Zetasizer, Malvern), (B) integrity (electron microscopy) and (C) sizedistribution (Nanosight, Malvern). No major changes with exception of aslight decrease in zeta potential are noted.

FIG. 13A-C. Electron microscopy validates the presence of hsiRNA at thesurface and in the lumen of exosomes. Exosomes were incubated withbiotinylated HTT10150 hsiRNA and purified as described herein. Electronmicroscopy was performed in the absence (data not shown) or presence (A)of streptavidin immunogold particles on sample not treated (A) ortreated with 0.1% saponin (B). (C) Quantification of gold particlesinside or outside of exosomes.

FIG. 14. Efficient internalization of hydrophobic Cy3-hsiRNA-loadedexosomes in primary cortical neurons. Cy3-HTT10150 hsiRNA-loadedexosomes (PKH67 labeled, green) were added to primary cortical neurons,and internalization was followed by imaging on Leica confocal microscope(63×). Nuclei were stained with Hoechst dye (blue). A significant levelof co-localization between exosomes and hsiRNA was observed.

FIG. 15A-C. Concentration dependent silencing of huntingtin mRNA byHTT10150-loaded exosomes in primary cortical neurons. Exosomes wereloaded with 20 μM hsiRNA HTT10150 or NTC as described herein. (A)Primary cortical neurons were incubated with non-loaded exosomes (leftbar; light grey), NTC-loaded exosomes (middle bar; dark grey) andHTT10150-loaded exosomes (right bar; black) for one week. The level ofhuntingtin mRNA was measured using QUANTIGENE assay (Affymetrix) at 7days and normalized to the housekeeping gene, PPIB (cyclophillin B), andpresented as percent of untreated control (n=3 replicates, mean±SD).NTC=non-targeting control. (B and C) Primary cortical neurons wereincubated with Cy3-hsiRNA-loaded exosomes for 4 days. (B) The level ofCy3 fluorescence was monitored in cell lysate by HPLC (n=3 replicates,mean±SD). (C) The proportion of Cy3 fluorescence detected in cell lysatewas calculated among the total fluorescence added on cells.

FIGS. 16 A and B. Bilateral distribution of Cy3-hsiRNA-loaded exosomesupon unilateral brain infusion. hsiRNA-loaded exosomes orCy3-hsiRNA-loaded exosomes (red) were loaded into an ALZET pump andunilaterally infused into the striatum of WT (FVBj) mice. Mice weresacrificed after 7 days. 40 μM coronal sections of the brain werestained with Dapi (cyan) as nuclear marker. (A) Pictures of the striatumand cortex were acquired with Leica DM5500-DFC365FX; 2.5×.Representative pictures are shown (n=2). (B) Magnifications wereacquired with Leica DM5500-DFC365FX; 63×. Representative pictures areshown (n=2 per group). Unilateral infusion resulted in bilateraldistribution of hsiRNA-loaded exosomes throughout the brain, withCy3-hsiRNA detectable in striatum and cortex on both sides of the brain.

FIG. 17. Bilateral uptake of HTT10150-loaded exosomes in neurons inmouse brain. hsiRNA-loaded exosomes or Cy3-hsiRNA-loaded exosomes (red)were loaded into an ALZET pump and unilaterally infused into thestriatum of WT (FVBj) mice. Mice were sacrificed after 7 days. 40 μMcoronal sections of the brain were stained with Dapi (blue) as a nuclearmarker and NeuN (green) as a neuron marker. Pictures of the striatumwere acquired with Zeiss confocal microscope; 63× oil objective.Representative pictures are shown (n=2). Confocal fluorescent microscopyof tissues co-stained with NeuN (neuronal marker) showed accumulation ofcompounds in neurons Arrows=accumulation of Cy3-hsiRNA in neurons.

FIGS. 18A and B. hsiRNA-loaded exosomes induce bilateral HTT mRNAsilencing in vivo in mouse brain. (A) ALZET pumps with PBS, aCSF,unloaded exosomes, HTT10150 (1 μg/day), NTC-loaded exosomes andHTT10150-loaded exosomes (2-4×1010 particle/day—hsiRNA 0.5 and 1 μg/dayfor 7 days) (n=10 for each group) were unilaterally implanted into theright striatum of WT (FVBNj) mice. Mice were sacrificed after 7 days.Brains were sliced into 300 μm sections and three 2 mm punch biopsies ofboth striatum were collected. The level of huntingtin mRNA was measuredusing QUANTIGENE assay (Affymetrix), normalized to housekeeping gene,PPIB (cyclophillin B), and presented as a percent of untreated control(n=30 punches, mean±SD). ** P<0.05, *** P<0.01, one-way ANOVA test,Bonferroni correction. aCSF=Artificial CerebroSpinal Fluid;CL=Contralateral; IL=Ipsilateral. (B) Table showing descriptivestatistics.

FIG. 19A-F. Exosomes and hsiRNA-loaded exosomes have no impact on immuneresponse in vivo. (A to C) PBS, exosomes or HTT10150-loaded exosomeswere unilaterally injected into the striatum of WT (FVBj) mice. Micewere perfused after 6 hours and brains were sliced into 40 μm sections.(A and B) Sections were stained with Iba1 antibody to evaluatemicroglial response. White arrowhead=resting microglia; Blackarrowhead=Activated microglia. (C) Quantification of resting andactivated microglia cells was performed manually and showed a slightincrease in activated microglia in all samples in the ipsilateral sideof the brain. (D to F) PBS, exosomes or HTT10150-loaded exosomes wereunilaterally infused into the striatum of WT (FVBj) mice. Mice wereperfused after 7 days and brains were sliced into 40 μm sections. (D andE) Sections were stained with Iba1 antibody to evaluate microglialresponse. White arrowhead=resting microglia; Black arrowhead=Activedmicroglia. (F) Quantification of resting and activated microglia cellswas performed manually and showed an increase in activated microglia inall samples in the ipsilateral side of the brain.

FIG. 20A-C. Exosomes and hsiRNA-loaded exosomes have no impact on cellviability in vivo. HTT10150 loaded exosomes were unilaterally injectedinto the striatum of WT (FVBj) mice. Mice were perfused after 7 days andbrains were sliced into 40 μm sections. (A and B) Sections were stainedwith DARPP32 antibody. (C) Quantification of DARPP32 labeled cells didnot show any change in the number of cells on the injected vsnon-injected side.

FIGS. 21A and B. Exosomes and hsiRNA-loaded exosomes have no impact onimmune response in vivo. PBS, exosomes and hsiRNA-loaded exosomes wereunilaterally injected into striatum or loaded into Alzet pump andunilaterally infused into the striatum of WT (FVBj) mice (n=5 pergroup). Mice were respectively sacrificed after 6 hours or 7 days. 40 μmcoronal sections of the brain were stained with Iba1 antibody toevaluate cell death. (A) Scans of brain section were acquired using theNikon CoolScan5000 slide scanner. (B) Magnifications of the ipsilateraland contralateral striatum were acquired with Leica DM5500-DFC365FX;63×. Representative pictures are shown. White arrowhead=restingmicroglia; Black arrowhead=Actived microglia. NTC=Non Targeting Control.

DETAILED DESCRIPTION OF THE INVENTION

One limitation to the wide-spread adoption of oligonucleotide-basedtherapies is the highly inefficient transit of oligonucleotides fromoutside cells to the intracellular compartments where functionalactivity of the oligonucleotides takes place. Exploiting natural,evolutionarily conserved, mechanisms and pathways for trafficking ofsmall RNAs across cellular boundaries may fundamentally improve theefficiency of oligonucleotide-based therapy. It has been previouslydemonstrated that exosomes might efficiently transfer therapeuticoligonucleotides to cells and tissues. However, one of the maintechnical unresolved issues is the loading of exosomes witholigonucleotides.

Current methods of loading exosomes with nucleic acid cargo includeelectroporation, or transfection with cationic lipid reagents. Othermethods include loading by ultracentrifugation. Each of these methodsresults in very low loading efficiency, where only a small fraction ofadded oligonucleotide molecules are transferred to exosomes. Forexample, one laboratory reported a loading efficiency usingultracentrifugation of 1 molecule of siRNA per 1000 exosomes.

New methods of loading exosomes with nucleic acid cargo are describedherein. These methods are based, at least in part, on the discovery thatintroduction of a hydrophobic modification into an oligonucleotidefacilitates exosomal loading. Accordingly, in some embodiments, thepresent invention relates to improved methods of loading exosomes withnucleic acid cargo. In other embodiments, the invention relates toexosomes loaded with nucleic acid cargo, in which the nucleic acidmolecules contain one or more hydrophobic modifications. In otherembodiments, the invention relates to the use of exosomes loaded withhydrophobically-modified nucleic acid cargo as delivery vehicles, e.g.,for delivery of small oligonucleotides to cells or tissues.

I. Definitions

Prior to setting forth the invention in detail, definitions of certainterms to be used herein are provided. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning as iscommonly understood by one of skill in the art.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising, “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value recited or falling within the range,unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recited.

The term “nucleotide” as used herein refers to a modified or naturallyoccurring deoxyribonucleotide or ribonucleotide capable of Watson-Crickbase pairing with a complementary nucleotide. Nucleotides can includenucleotide analogs (e.g., morpholinos, PMOs, etc.) capable ofWatson-Crick base pairing. Nucleotides typically include purines andpyrimidines, which include thymidine, cytidine, guanosine, adenine anduridine.

The term “oligonucleotide” or “nucleic acid molecule” as used hereinrefers to an oligomer of the nucleotides defined above.

The term “hydrophobic modification” as used herein refers to amodification that increases the hydrophobicity of an oligonucleotide, ascompared to native (non-modified) RNA or DNA.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein can be modified by theterm about.

The recitation of an embodiment for a variable or aspect herein includesthat embodiment as any single embodiment or in combination with anyother embodiments or portions thereof.

As will be evident to one of ordinary skill, any compositions or methodsprovided herein can be combined with one or more of any of the othercompositions and methods provided herein.

II. Exosomes

Exosomes are small vesicles that originate in eukaryotic cells,primarily from the endosomal pathway. Exosomes are bound by a plasmamembrane and are released from cells into the extracellular environment.Generally, these vesicles are approximately 30-100 nM in diameter, butcan range in size from approximately 20 nM to approximately 200 nM.Naturally occurring exosomes are hypothesized to transport moleculesfrom one cell to another. Exosomes are taken up by recipient cells byendocytosis or by fusion of the exosomal membrane with the plasmamembrane of the recipient cell.

These and other properties of exosomes have led to their use as deliveryvehicles for synthetic cargo, e.g., proteins and nucleic acids. Exosomesare an attractive alternative to liposomes for use as delivery vehicles,because they readily cross major biological membranes due to their smallsize and the nature of their lipid bi-layer. They are well-tolerated bysubjects, and are highly stable in biological fluids, which protectsexosomal cargo from degradation.

Exosomes can be isolated and/or purified from the media of cells grownin culture (i.e., conditioned media), or from biological fluids obtainedfrom a subject, e.g., plasma, blood, urine, lymph, etc. A majority ofcell types produce exosomes, including, but not limited to, dendriticcells (DC), B cells, T cells, mast cells, epithelial cells, stem cells,neuronal cells, and tumor cells. Accordingly, exosomes used in themethods and compositions described herein can be derived from any ofthese cell types, or combinations thereof. By way of example, exosomescan be derived from immune cells, B lymphocytes, T lymphocytes,dendritic cells, immature dendritic cells, mast cells, neuronal cells,stem cells and/or tumor cells. In some embodiments, exosomes are derivedfrom immature dendritic cells that do not express MHC-I, MHC-II, orCD86, and consequently minimize clearance by the immune system of asubject following administration. In an exemplary embodiment, exosomesare derived from neuronal cells. In another exemplary embodiment,exosomes are derived from stem cells, for example, adult pluripotentstem cells. In certain embodiments, exosomes are derived from inducedpluripotent stem cells (iPS cells). In another exemplary embodiment,exosomes are derived from U87 cells.

Exosomes can be isolated from cells grown in culture or from biologicalfluids by any suitable method known in the art. For example, exosomescan be isolated using techniques including differential centrifugation,precipitation, gel-filtration, column binding, affinity purification, orcombinations of these methods. By way of non-limiting example, exosomescan be purified by isolation from cells and other cellular components bydifferential centrifugation, whereby cell culture supernatants arecentrifuged at low speeds (e.g., 20,000 g or less) to remove cells andcellular debris, followed by centrifugation at high speeds (e.g.,100,000 g or more) to pellet exosomes. This procedure can be used inconjunction with filtration (e.g., using filters of approximately 0.8 μMand/or 0.2 μM) to eliminate cell debris and other contaminants. Inanother example, exosomes can be purified using a density gradient,e.g., a sucrose density gradient, to isolate exosomes having an averagedensity of approximately 1.13-2.21 g/mL. In an exemplary embodiment,exosomes can be purified by centrifugation at approximately 300 g toremove cells, followed by centrifugation at approximately 2000 g toremove dead cells, followed by centrifugation at approximately 10,000 gto remove cellular debris. The supernatant can be filtered using afilter of, for example, approximately 0.2 μm. The filtrate can then becentrifuged at 100,000 g to pellet exosomes. If desired, the pellet canbe washed, and centrifuged again at 100,000 g to further purify theexosome population. Exemplary protocols for isolating exosomes aredescribed in FIG. 1A and FIG. 9A.

Other methods of exosomal isolation are described by Raposo et al.(1996), B lymphocytes secrete antigen-presenting vesicles, J. Exp. Med.183:1161-1172. One exemplary method of purifying exosomes from a largevolume of conditioned medium by ultrafiltration is described byLamparski et al. (2002), Production and characterization of clinicalgrade exosomes derived from dendritic cells, J. Immunol. Methods270:211-226. This method is particularly for isolation of exosomes fortherapeutic administration to a subject. Other exemplary methods ofexosome purification are described by Thery et al., Isolation andCharacterization of Exosomes from Cell Culture Supernatants andBiological Fluids, Current Protocols in Cell Biology (2006)3.22.1-3.22.29.

A number of kits are commercially available to facilitate purificationof exosomes from a variety of source material. These include, forexample, the Exo-spin™ Exosome Purification Kit from Cell GuidanceSystems, the Total Exosome Isolation Kit from Life Technologies, theExoQuick™ Exosome Isolation Kit from System Biosciences, and theExo-Flow™ exosome immunopurification kit from System Biosciences.

In some embodiments, exosomes are synthetically produced. In suchembodiments, artificial exosomes can be created by embedding exosomalproteins in a lipid bilayer. For example, liposomes can be assembledwhich contain a variety of components found in native exosomes. Suchsynthetic exosomes are sometimes termed exosome mimetics. Syntheticexosomes are described in, for example, Kooijmans et al., Int. J.Nanomedicine (2012), 7:1525-1541, and in De La Pena et al., J. Immunol.Methods (2009), 344(2):121-32.

III. Loading Exosomes with Hydrophobically Modified Nucleic Acid Cargo

In certain embodiments, the invention relates to a highly efficientmethod of loading exosomes with nucleic acid cargo. Currently, apredominant obstacle to the commercialization of exosomes as a deliveryvehicle for oligonucleotides is highly inefficient loading. Thisobstacle can be overcome by hydrophobically modifying nucleic acid cargoprior to loading the cargo into exosomes. As described herein,hydrophobic modification of nucleic acid cargo facilitates loading ofnucleic acid into exosomes. Without wishing to be bound by theory, it isproposed that hydrophobic modification of nucleic acid cargo allowsself-assembly of the cargo into exosomal vesicles. Surprisingly,hydrophobic modification of nucleic acid cargo permits exosomal loadingin the absence of electroporation, and without the use of transfectionreagents, e.g., cationic liposome transfection reagents. Hydrophobicmodification of nucleic acid cargo also permits exosomal loading withoutthe need for ultracentrifugation (however, in some embodiments,ultracentrifugation may nonetheless be useful for purification ofexosomes prior to or after loading). Hydrophobically modified nucleicacid cargo can be loaded into exosomes with significantly improvedefficiency relative to that which is generally reported for methods ofloading exosomes by traditional methods, for example, electroporation,lipid-mediated transfection, or ultracentrifugation.

Accordingly, in some embodiments, the invention features a method ofloading exosomes with oligonucleotide cargo, by incubating ahydrophobically modified oligonucleotide with a population of exosomesfor a period of time sufficient to permit loading of the exosomes withthe hydrophobically modified oligonucleotide.

In other embodiments, the invention features a method of loadingexosomes with oligonucleotide cargo, consisting of or consistingessentially of incubating a hydrophobically modified oligonucleotidewith a population of exosomes for a period of time sufficient to permitloading of the exosomes with the hydrophobically modifiedoligonucleotide.

In other embodiments, the invention features a method of loadingexosomes with oligonucleotide cargo, by introducing one or morehydrophobic modifications into the oligonucleotide cargo, and incubatingthe hydrophobically modified oligonucleotide with a population ofexosomes for a period of time sufficient to permit loading of theexosomes with the hydrophobically modified oligonucleotide.

In a preferred aspect of each of the foregoing embodiments, exosomes areloaded without the use of electroporation, and in the absence oftransfection reagents. For example, exosomes can be efficiently loadedwith hydrophobically modified oligonucleotide in the absence oflipid-based transfection reagents, such as cationic liposometransfection reagents. Exosomes can also be loaded without the use ofultracentrifugation (however, in some embodiments, ultracentrifugationmay nonetheless be useful for purification of exosomes prior to or afterloading).

The duration of time sufficient to permit loading of the exosomes withhydrophobically modified oligonucleotide cargo can be optimized for theparticular type of cargo and the type of modification. Generally, anincubation of 1 hour or less is sufficient to permit efficient loadingof exosomes with hydrophobically modified cargo. In many instances,hydrophobically modified cargo is efficiently loaded into exosomes in avery rapid period of time, for example, within 5 minutes. Accordingly,in some embodiments, efficient loading takes place during an incubationperiod of 5 minutes or less, e.g., from 1-5 minutes. In exemplaryembodiments, efficient loading takes place during an incubation periodof 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, etc. Inother embodiments, efficient loading may take place within 1 hour,within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours,within 12 hours, within 24 hours, etc.

Loading of exosomes with hydrophobically modified oligonucleotides isnot highly temperature dependent. In exemplary embodiments, exosomes areloaded at or around 37° C. In other embodiments, exosomes can be loadedat or around room temperature. In other embodiments, exosomes can beloaded at or around 4° C.

The methods of loading exosomes with oligonucleotide cargo (i.e.hydrophobically modified oligonucleotide cargo) set forth hereinsignificantly improve loading efficiency as compared to the loadingefficiency previously reported for introducing unmodified nucleic acidcargo into exosomes by, for example, electroporation or cationic lipidtransfection. In some embodiments, over 50% of hydrophobically modifiedoligonucleotide cargo is incorporated into exosomes using the methodsdescribed herein. Accordingly, in some embodiments, hydrophobicallymodified oligonucleotide cargo is incorporated into exosomes with anefficiency of 5-40%. For example, hydrophobically modifiedoligonucleotide cargo is incorporated into exosomes with an efficiencyof 5% or greater, 10% or greater, 15% or greater, 20% or greater, 25% orgreater, 30% or greater, 35% or greater, 40% or greater, or 50% orgreater. The methods described herein result in the incorporation ofhydrophobically modified oligonucleotide into all or nearly all of theexosomes that are treated. For example, at least 80% of the exosomesincubated with hydrophobically modified oligonucleotide are typicallyloaded with the oligonucleotide. In some embodiments, hydrophobicallymodified oligonucleotide cargo is incorporated in at least 90% of theexosomes incubated with the oligonucleotide. Thus, populations ofexosomes in which at least 80%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98% or more of the exosomes are loaded with theoligonucleotide cargo can be readily obtained. In one embodiment atleast 99% of the exosomes are loaded with the hydrophobically modifiedoligonucleotide.

The methods described herein also allow greater quantities of cargooligonucleotides to be loaded into exosomes than could be achieved usingtraditional methods. For example, exosomes can be loaded with over 500hydrophobically modified oligonucleotide molecules per exosome, e.g., atleast 500, at least 600, at least 700, at least 800, at least 900, atleast 1000, at least 1200, at least 1500, at least 2000, at least 2500,at least 3000 or more hydrophobically modified oligonucleotides perexosome. In one embodiment, the exosomes contain an average of about500-3000 hydrophobically modified oligonucleotides per exosome. Inanother embodiment, exosomes contain an average of about 500-1000hydrophobically modified oligonucleotides per exosome. In anotherembodiment, exosomes contain an average of about 1000-1500hydrophobically modified oligonucleotides per exosome. In anotherembodiment, exosomes contain an average of about 1000-2000hydrophobically modified oligonucleotides per exosome. In anotherembodiment, exosomes contain an average of about 1000-3000hydrophobically modified oligonucleotides per exosome. In anotherembodiment, exosomes contain up to about 3000 hydrophobically modifiedoligonucleotides per exosome. The quantities of hydrophobically modifiedoligonucleotide cargo that can be loaded into exosomes allow theproduction of exosomes in which the hydrophobically modifiedoligonucleotide cargo occupies a significant proportion of the exosomalmembrane. For example, exosomes can be produced in which hydrophobicallymodified oligonucleotide cargo occupies about 1-10% of the surface areaof the exosome, e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%of the surface area of the exosome.

In another embodiment, hydrophobically modified nucleic acid moleculescan be incorporated into exosomes during exosome production by cells inculture. In accordance with this embodiment, hydrophobically modifiednucleic acid molecules can be incubated with cells in culture, resultingin efficient uptake of hydrophobic nucleic acid by cells. Cells are thenincubated for a period of time sufficient for exosome production.Exosomes isolated from the culture media will contain exosomes loadedwith the hydrophobic nucleic acid molecule taken up by the cells.Accordingly, in one embodiment, a method of loading exosomes witholigonucleotide cargo is provided, comprising incubating cells capableof exosome production with an oligonucleotide containing one or morehydrophobic modifications for a period of time sufficient for theoligonucleotide to be internalized by the cells, culturing the cells fora period of time sufficient for exosome secretion, and isolatingexosomes loaded with the oligonucleotide from the culture medium.

IV. Hydrophobically Modified Nucleic Acid Molecules

Oligonucleotide molecules which are amenable to hydrophobic modificationcan be loaded into exosomes as described herein. In some embodiments,the oligonucleotide cargo is DNA. In other embodiments, theoligonucleotide cargo is RNA. In other embodiments, the oligonucleotidesare nucleotide analogs. Non-limiting examples of oligonucleotidemolecules which can be loaded into exosomes as described herein includesiRNA, siRNA-GalNAc, antisense, Locked Nucleic Acids (LNAs), hairpinsiRNA, phosphorodiamidate morpholino oligomers (PMOs), miRNA, andoligonucleotide miRNA inhibitors. In some embodiments, theoligonucleotide molecules are plasmid DNA, which can be modified with ahydrophobic modification post-transcriptionally. In an exemplaryembodiment, the oligonucleotide cargo is a siRNA. In another exemplaryembodiment, the oligonucleotide cargo is a hairpin siRNA. In anotherexemplary embodiment, the oligonucleotide cargo is a miRNA.

In certain embodiments, the oligonucleotide cargo is capable ofmodifying gene expression in a target cell. For example, theoligonucleotide cargo may reduce or inhibit expression of one or moregenes in a target cell. This can occur by way of direct targeting of DNAor RNA through Watson-Crick base pairing. By way of example, cargomolecules capable of reducing or inhibiting expression of one or moregenes in a target cell can include siRNA, siRNA-GalNAc, antisense,Locked Nucleic Acids (LNAs), hairpin siRNA, phosphorodiamidatemorpholino oligomers (PMOs), miRNA, and oligonucleotide miRNAinhibitors. In other embodiments, the oligonucleotide cargo may increaseexpression of one or more genes in a target cell.

By way of example, cargo molecules capable of increasing expression ofone or more genes in a target cell include expression vectors andoligonucleotide miRNA inhibitors.

In some embodiments, the oligonucleotide cargo is a therapeuticoligonucleotide. A therapeutic oligonucleotide is useful in treating orameliorating the signs and symptoms of a disease or disorder whenadministered to a subject. For example, a therapeutic oligonucleotidecan target a gene involved in a disease process, thereby reducing thesymptoms of the disease in a subject to whom the therapeuticoligonucleotide is administered.

In order to facilitate exosomal loading, oligonucleotide cargo containsone or more hydrophobic modifications. Hydrophobic modificationsincrease the hydrophobicity of the oligonucleotide cargo, as compared tonative (non-modified) RNA or DNA. In certain embodiments, thehydrophobic modifications increase the hydrophobicity of theoligonucleotide by at least two orders of magnitude (e.g., at least 3,4, 5, 6, 7, 8, 9, 10 or more orders of magnitude) relative to native(non-modified) RNA or DNA. In other embodiments, the hydrophobicmodifications increase the hydrophobicity of the oligonucleotide by atleast 10 orders of magnitude relative to native (non-modified) RNA orDNA. In other embodiments, the hydrophobic modifications increase thehydrophobicity of the oligonucleotide by at least two orders ofmagnitude (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more orders ofmagnitude) relative to the unmodified oligonucleotide. In otherembodiments, the hydrophobic modifications increase the hydrophobicityof the oligonucleotide by at least ten orders of magnitude relative tothe unmodified oligonucleotide. Increases in hydrophobicity can beassessed using any suitable method. For example, hydrophobicity can bedetermined by measuring the percentage solubility in an organic solvent,such as octanol, as compared to solubility in an aqueous solvent, suchas water.

In some embodiments, the hydrophobic character of oligonucleotide cargocan be increased by increasing the proportion of nucleotides within theoligonucleotide molecule that are hydrophobically modified. For example,in one embodiment, 20% or more of the nucleotides in an oligonucleotidemolecule are hydrophobically modified, e.g., 25% or more, 30% or more,40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% ormore, 95% or more, 99% or more, etc. of the nucleotides in anoligonucleotide molecule are hydrophobically modified. In oneembodiment, 100% of the nucleotides in an oligonucleotide molecule arehydrophobically modified. In an exemplary embodiment, 30% or more of thenucleotides in an oligonucleotide molecule contain hydrophobicmodifications. Increasing the proportion of hydrophobically modifiednucleotides in an oligonucleotide molecule can be useful when, forexample, the hydrophobic modification is weakly hydrophobic, forexample, a 2′O-methyl modification. In embodiments where a stronglyhydrophobic modification is employed, for example, a sterol, a lipid,etc., a single hydrophobic modification can be sufficient to facilitateexosomal loading.

In preferred embodiments, the hydrophobic modification is a covalentmodification. Exemplary hydrophobic modifications are depicted in FIG. 3and FIG. 10, and in Table 2.

Hydrophobic modifications of nucleic acid molecules can include, forexample, backbone modifications, sugar modifications, base modificationsand/or conjugate modifications, and combinations thereof.

Backbone modifications involve alterations to the phosphate esterlinkages in the nucleic acid molecule. Examples of suitable backbonemodifications include, but are not limited to, phosphorothioatemodifications, phosphorodithioate modifications, p-ethoxy modifications,methylphosphonate modifications, methylphosphorothioate modifications,alkyl- and aryl-phosphates (in which the charged phosphonate oxygen isreplaced by an alkyl or aryl group), alkylphosphotriesters (in which thecharged oxygen moiety is alkylated), peptide nucleic acid (PNA) backbonemodifications, locked nucleic acid (LNA) backbone modifications, and thelike. These modifications may be used in combination with each otherand/or in combination with phosphodiester backbone linkages.

In one embodiment, the hydrophobic modification is a phosphorothioate(PS) modification, where one of the nonbridging phosphate oxygen atomsis replaced by sulfur to give a PS group (see, for example, Eckstein,Biochimie. 2002, 84, 841-848). This modification provides significantresistance to nuclease degradation and has favorable pharmacokineticproperties (Bumcrot et al, Nat. Chem. Biol. 2006, 2, 711-719). PSlinkages can readily incorporated into oligonucleotide molecules usingstandard techniques, such as solid-phase oligonucleotide synthesis(Sanghvi, Current Protocols in Nucleic Acid Chemistry, 2011,4.1.1-4.1.22).

In another embodiment, the hydrophobic modification is a phosphonatemodification, in which one nonbridging oxygen is replaced with an alkylgroup. In other embodiments, the hydrophobic modification is a peptidenucleic acid (PNA) modification. PNAs are oligonucleotide mimics thathave a peptide backbone with a neutral charge, as compared with thehighly charged sugar-phosphate backbone of native RNA and DNA (see, forexample, Nielsen et al, Science 1991, 254, 1497-1500; Demidov et al,Biochem Pharmacol, 1994, 48, 1310-1313). In other embodiments, thehydrophobically modified nucleic acid molecule is a phosphorodiamidatemorpholino oligonucleotide (PMO).

In other embodiments, oligonucleotide cargo molecules may behydrophobically modified at the sugar moiety (e.g., ribose, deoxyribose,etc.). Sugar modifications often occur at the 2′ position of the sugarring, where, for example, the 2′ moiety can be modified or substitutedwith a hydrophobic moiety, such as a halo, alkoxy, aminoalkoxy, alkyl,azido or amino group. In non-limiting examples, sugar modifications caninclude O-methyl, F, methoxy-ethyl, and 2′-fluoro-β-D-arabinonucleotide(FANA). Other 2′ modifications include, for example, 2′O-allyl,2′O-ethylamine, and 2′O-cyanoethyl modifications. In addition,modifications can be made at other sites including the 4′ position ofthe sugar (see, for example, Deleavey, et al, Chem Bio, 2012, 19,937-954).

In other embodiments, oligonucleotide cargo molecules may containhydrophobic base modifications. In exemplary embodiments, thesemodifications include phenyl, naphthyl, and isobutyl. Other embodimentsinclude C-5 propynyl modified bases, 5-methylcytosine, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, and hypoxanthine.

In addition to increasing the hydrophobic character of theoligonucleotide cargo, the foregoing backbone, sugar, and basemodifications increase the stability of the olignoucleotides in thepresence of exosomes, and minimize any degradation that may occur duringloading.

Hydrophobic moieties can also be chemically conjugated tooligonucleotide cargo to enhance its hydrophobic character. In exemplaryembodiments, the moiety is a sterol (e.g., cholesterol), GM1, a lipid, avitamin, a small molecule, a peptide, or a combination thereof. In someembodiments, the moiety is a lipid. For example, in certain embodiments,the moiety is palmitoyl. In some embodiments, the moiety is a sterol,e.g., cholesterol. Additional hydrophobic moieties include, for example,phospholipids, vitamin D, vitamin E, squalene, and fatty acids. Inanother exemplary embodiment, the oligonucleotide cargo is conjugated tomyristic acid, or a derivative thereof (e.g., myristoylatedoligonucleotide cargo). In some embodiments, the hydrophobic moiety isconjugated at the termini of the oligonucleotide cargo (i.e., “terminalmodification”). In other embodiments, the hydrophobic moiety isconjugated to other portions of the oligonucleotide molecule.

In one embodiment, the oligonucleotide cargo is stabilized byincorporation of one or more backbone modifications, sugarmodifications, and/or base modifications as described herein, andadditionally is conjugated to a hydrophobic moiety. Exemplaryembodiments are shown in FIG. 3 and FIG. 10. For example, theoligonucleotide cargo in certain embodiments can contain one or morebackbone modifications, sugar modifications, and/or base modificationsto at least 30%, at least 35%, at least 40%, at least 45%, at least 50%,at least 55%, at least 60%, at least 65% or more of the nucleotides, andfurther is conjugated to a hydrophobic moiety as described herein, e.g.,conjugated to a sterol, GM1, a lipid, a vitamin, a small molecule, or apeptide, or a combination thereof. In an exemplary embodiment, theoligonucleotide cargo is conjugated to a sterol, e.g., cholesterol. Inanother exemplary embodiment, the oligonucleotide cargo is conjugated toGM1. In another exemplary embodiment, the oligonucleotide cargo isconjugated to myristic acid, or a derivative thereof.

In one embodiment, the oligonucleotide cargo is an siRNA that contains ashort duplex region (for example, 14-16 base pairs, e.g., 15 basepairs), and a single-stranded fully phosphorothioated tail. Thisembodiment is illustrated in FIG. 10. In this exemplary construct,pyrimidines are modified with 2′-fluoro and 2′-O-methyl modifications.The 3′ end of the passenger strand of this exemplary construct isconjugated to cholesterol.

In some embodiments, the hydrophobically modified oligonucleotide caninclude a detectable label. Exemplary labels include fluorescent labelsand/or radioactive labels. In embodiments where hydrophobically modifiedoligonucleotides are fluorescently labeled, the detectable label can be,for example, Cy3. Adding a detectable label to hydrophobically modifiedoligonucleotides can be used as a way of labeling exosomes, andfollowing their biodistribution. In other embodiments, a detectablelabel can be attached to exosomes directly, for example, by way oflabeling an exosomal lipid and/or an exosomal peptide.

Nucleic acids can be synthesized using any number of procedures known inthe art. A number of automated nucleic acid synthesizers arecommercially available for this purpose. In a preferred embodiment, thenucleic acid cargo is a synthetic oligonucleotide. In other embodiments,nucleic acids can be prepared using, for example, restriction enzymes,exonucleases, or endonucleases.

V. Compositions Containing Exosomes Loaded with Hydrophobically ModifiedCamp

In certain aspects, the invention provides exosomes loaded withhydrophobically modified oligonucleotide cargo. For example, in oneaspect, the invention provides a composition comprising a population ofexosomes loaded with an oligonucleotide comprising one or morehydrophobic modifications. Exemplary exosome populations andhydrophobically modified oligonucleotides are set forth herein.

For example, the invention includes, in various embodiments, exosomesloaded with the hydrophobically modified oligonucleotides describedherein. By way of example, the hydrophobically modified oligonucleotidescan be synthetic oligonucleotides. In some embodiments, thehydrophobically modified oligonucleotides are siRNA, siRNA-GalNAc,antisense RNA, LNA, hairpin siRNA, PMO, miRNA, miRNA inhibitors, orcombinations thereof. In exemplary embodiments, the hydrophobicallymodified oligonucleotides are siRNA or miRNA.

In some embodiments, the hydrophobic modifications increase thehydrophobicity of the oligonucleotide by at least two orders ofmagnitude, e.g., 2-10 orders of magnitude, as described above. In someembodiments, the hydrophobic character and/or the stability of theoligonucleotide cargo can be increased by increasing the proportion ofnucleotides within the oligonucleotide molecule that are hydrophobicallymodified, as described above. Exemplary hydrophobic modificationsinclude, e.g., backbone modifications, ribose modifications, basemodifications, and combinations thereof, as described herein. Forexample, in embodiments the oligonucleotide cargo contains one or morebackbone modifications, sugar modifications, and/or base modificationsto at least 30%, at least 35%, at least 40%, at least 45%, at least 50%,at least 55%, at least 60%, at least 65% or more of the nucleotides Insome embodiments, the oligonucleotide is conjugated to a hydrophobicmoiety, e.g., sterol, GM1, a lipid, a vitamin, a small molecule, or apeptide, or combinations thereof. Other examples of hydrophobic moietiesare described herein, and include but are not limited to cholesterol,GM1, and myristic acid, or a derivative thereof.

In some embodiments, at least 80% of the exosomes in the compositions ofthe invention are loaded with the hydrophobically modifiedoligonucleotide. In a preferred embodiment, at least 90% of the exosomesare loaded with the hydrophobically modified oligonucleotide. Inexemplary embodiments, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% ormore of the exosomes are loaded with the hydrophobically modifiedoligonucleotide. In one embodiment at least 99% of the exosomes areloaded with the hydrophobically modified oligonucleotide.

In other embodiments, the compositions of the invention contain exosomesthat are loaded with an average of at least 500 hydrophobically modifiedoligonucleotides per exosome, e.g., at least 600, at least 700, at least800, at least 900, at least 1000, at least 1200, at least 1500, at least2000, at least 2500, at least 3000 or more hydrophobically modifiedoligonucleotides per exosome. In one embodiment, the exosomes contain anaverage of about 500-3000 hydrophobically modified oligonucleotides perexosome. In another embodiment, exosomes contain an average of about500-1000 hydrophobically modified oligonucleotides per exosome. Inanother embodiment, exosomes contain an average of about 1000-1500hydrophobically modified oligonucleotides per exosome. In anotherembodiment, exosomes contain an average of about 1000-3000hydrophobically modified oligonucleotides per exosome. In anotherembodiment, exosomes contain up to about 3000 hydrophobically modifiedoligonucleotides per exosome.

In another embodiment, the compositions of the invention include aplurality of exosomes in which hydrophobically modified oligonucleotidecargo occupies about 1-10% of the exosome surface, e.g., about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the surface area of the exosome.

Exosomes suitable for use in the compositions of the invention includeexosomes having the characteristics described herein. For example, inone embodiment, the exosomes are derived from cultured cells, includingbut not limited to dendritic cells (DC), B cells, T cells, mast cells,epithelial cells, stem cells, neuronal cells, and tumor cells. In someembodiments, the cultured cells are immature dendritic cells, neuronalcells, or stem cells, e.g., iPS cells. In other embodiments, theexosomes are synthetic exosomes. Optionally, the exosomes can include atargeting peptide, which can facilitate targeting of the exosomes to aparticular cell type, for example, neuronal cells.

In some embodiments, the exosome compositions are pharmaceuticalcompositions. Accordingly, in one aspect, the invention providespharmaceutical compositions containing exosomes loaded withhydrophobically modified oligonucleotides, as described herein, and apharmaceutically acceptable carrier or excipient. Such compositions caninclude exosomes loaded with a therapeutically effective amount of anoligonucleotide comprising one or more hydrophobic modifications, and apharmaceutically acceptable carrier or excipient. Such pharmaceuticalcompositions are suitable for administration to a subject, e.g. a humansubject or an animal subject. Pharmaceutical compositions containingexosomes loaded with hydrophobically modified oligonucleotides aredescribed in additional detail below.

The simplicity and scalability of the exosomal loading methods describedherein allow oligonucleotide-loaded exosomes to be readily generated insufficient quantities for administration to a subject. In oneembodiment, the pharmaceutical compositions contain at least about 10⁷exosomes, e.g., at least about 10⁸ exosomes, about 10⁹ exosomes, about10¹⁰ exosomes, about 10¹¹ exosomes, about 10¹² exosomes, about 10¹³exosomes, about 10¹⁴ exosomes, about 10¹⁵ exosomes, about 10¹⁶ exosomes,about 10¹⁷ exosomes, about 10¹⁸ exosomes, or about 10¹⁹ exosomes. In anexemplary embodiments, the pharmaceutical compositions contain about10⁸-10¹⁵ exosomes. Additional dosage amounts of exosomes suitable fortherapeutic administration are described below.

In some embodiments, at least about 90% of the exosomes in thecomposition are loaded with hydrophobically modified oligonucleotide. Inother embodiments, at least about 99% of the exosomes in the compositionare loaded with hydrophobically modified oligonucleotide. The exosomesin the pharmaceutical compositions can be loaded with thehydrophobically modified oligonucleotide at concentrations describedherein. For example, the exosomes may contain an average of about500-1000 oligonucleotide molecules per exosome. In other embodiments,the exosomes may contain an average of about 1000-3000 oligonucleotidemolecules per exosome.

In another embodiment, the invention provides a pharmaceuticalcomposition containing a plurality of exosomes loaded with ahydrophobically modified oligonucleotide, as set forth in anyembodiments described herein, and a pharmaceutically acceptable carrieror excipient.

The composition may be formulated for parenteral, intramuscular,intracerebral, intravascular (including intravenous), subcutaneous, ortransdermal administration. Pharmaceutical compositions may includesterile aqueous solutions which may also contain buffers, diluents andother suitable additives. The compositions may include pharmaceuticallyacceptable carriers, thickeners, diluents, buffers, preservatives, andother pharmaceutically acceptable carriers or excipients and the like inaddition to the exosomes.

A “pharmaceutically acceptable carrier” (excipient) is apharmaceutically acceptable solvent, suspending agent or any otherpharmacologically inert vehicle for delivering exosomes to a subject.Typical pharmaceutically acceptable carriers include, but are notlimited to, binding agents (e.g., pregelatinised maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodiumstarch glycolate, etc.); or wetting agents (e.g., sodium laurylsulphate, etc.).

The compositions provided herein may additionally contain other adjunctcomponents conventionally found in pharmaceutical compositions. Thus,for example, the compositions may contain additional compatiblepharmaceutically-active materials or may contain additional materialsuseful in physically formulating various dosage forms of the compositionof present invention, such as dyes, flavouring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions providedherein.

In certain embodiments, the exosome compositions of the invention areproduced in accordance with the methods described herein. For example,exosome compositions containing exosomes loaded with hydrophobicallymodified oligonucleotides can be produced by incubating anoligonucleotide comprising one or more hydrophobic modifications with apopulation of exosomes.

VI. Exosome Delivery and Methods of Treatment

The exosomal compositions described herein can be used to deliveroligonucleotide cargo to cells. In one embodiment, the compositionsdescribed herein are used to deliver oligonucleotide cargo to cells inculture. In other embodiments, the compositions described herein areused to deliver oligonucleotide cargo to cells in a subject, i.e., ahuman or animal subject. Accordingly, in some embodiments, thecompositions described herein can be used to alter gene expression incells or tissues in vitro and/or in vivo by efficient delivery ofoligonucleotide cargo to such cells or tissues.

In some embodiments, exosomes can be specifically targeted to a desiredcell type or tissue type, e.g., damaged or diseased tissues. Forexample, exosomes can be specifically targeted to a desired cell type ortissue type by expression of a targeting peptide on the exosome surface.The targeting peptide can bind to a moiety present on the surface of thedesired target cells. For example, expression of a specific cell-surfacemarker can be induced on the exosome, which results in specificinteraction with a receptor on a desired target tissue.

Suitable targeting peptides are those which bind to cell surfacemoieties, such as receptors or their ligands, found on the cell surfaceof the cell to be targeted. Examples of suitable targeting moieties areshort peptides, scFv and complete proteins, so long as the targetingpeptide can be expressed on the surface of the exosome. In someembodiments, targeting peptides are full-length proteins. In otherembodiments, targeting peptides are fragments of full-length proteins.In some examples, targeting peptides are than 100 amino acids in length,for example less than 50 amino acids in length, less than 30 amino acidsin length, to a minimum length of 10, 5 or 3 amino acids.

Targeting peptides can be selected to target particular tissue typessuch as muscle, brain, liver, pancreas and lung for example, or totarget a diseased tissue such as a tumor. In a preferred embodiment, theexosomes are targeted to brain tissue. This can be achieved using atargeting peptide that interacts with a neuronal cell surface marker.Exemplary neuronal cell surface markers include, but are not limited toGM1, Neun etc. RVG peptides and the peptide part of tetanus and choleratoxin can also be used in some embodiments to specifically targetexosomes to neurons.

The targeting peptide targeting can be localized to the surface of theexosome by expressing it as a fusion protein with an exosomaltransmembrane protein. A number of proteins are known to be associatedwith exosomes. Exemplary exosomal transmembrane proteins include but arenot limited to Lamp-1, Lamp-2, CD13, CD86, Flotillin, Syntaxin-3, CD2,CD36, CD40, CD40L, CD41a, CD44, CD45, ICAM-I, Integrin alpha4, LiCAM,LFA-I, Mac-1 alpha and beta, Vti-1 A and B, CD3 epsilon and zeta, CD9,CD18, CD37, CD53, CD63, CD81, CD82, CXCR4, FcR, GluR2/3, HLA-DM (MHCII), immunoglobulins, MHC-I or MHC-II components, TCR beta andtetraspanins. In certain embodiments, the exosomal transmembrane proteinis Lamp-1, Lamp-2, CD13, CD86, Flotillin, or Syntaxin-3. In an exemplaryembodiment, the exosomal transmembrane protein is Lamp-2.

The exosomal transmembrane protein can be modified to incorporate atargeting moiety. For example, the exosomal transmembrane protein can beexpressed as a fusion protein comprising the targeting peptide. Thetargeting peptide is incorporated into the transmembrane protein suchthat it will be positioned on the surface of the exosomes. The targetingpeptide can be introduced into the exosome by expressing a fusionprotein comprising the targeting peptide and the exosomal transmembraneprotein (or a portion thereof) within a cell used to produce theexosomes. Expression of this fusion protein in the cell allows for thefusion protein to be incorporated into the exosome as it is producedfrom the cell. This process is described in, for example,US2013/0053426, the entire contents of which are incorporated herein byreference.

Antibodies and antibody fragments, e.g., scFv antibody fragments, canalso be used as targeting peptides to target specific antigens, such asNGFR for neuronal targeting. In addition, natural ligands for receptorscan be expressed as fusion proteins to promote exosome targeting. By wayof example, NGF and fragments thereof binds NGFR and thereby confersneuron-specific targeting.

In other embodiments, targeting of the exosomes is not performed.

In one embodiment, the native contents of the exosome are removed andreplaced with desired exogenous proteins or nucleic acids. In anotherembodiment, the native contents of exosomes are supplemented withdesired exogenous proteins or nucleic acids.

Exosomes loaded with hydrophobically modified oligonucleotide cargo canbe used therapeutically in subjects, i.e., human subjects or animalsubjects. Accordingly, in one aspect, the invention features a method oftreating a disease or disorder in a subject, by administering to thesubject a composition comprising exosomes loaded with hydrophobicallymodified oligonucleotide cargo. Any of the hydrophobically modifiedoligonucleotide molecules described herein are suitable cargo for suchmethods of treatment. Likewise, any of the exosomal preparationsdescribed herein are suitable for use in methods of treatment. Theparticular oligonucleotide cargo can be selected based upon the diseaseor disorder to be treated. Non-limiting examples of oligonucleotidemolecules which can be utilized in methods of treatment when loaded intoexosomes as described herein include siRNA, siRNA-GalNAc, antisense,Locked Nucleic Acids (LNAs), hairpin siRNA, phosphorodiamidatemorpholino oligomers (PMOs), miRNA, and oligonucleotide miRNAinhibitors. In some embodiments, the oligonucleotide cargo is an siRNA,a hairpin RNA, or a miRNA.

In some embodiments, hydrophobically modified oligonucleotide cargo isselected that reduces or inhibits expression of a gene associated with adisease or disorder.

For example, in a method of treating Huntington's disease,hydrophobically modified oligonucleotides targeting a mutant allele ofthe huntingtin gene are incorporated into the exosomes that areadministered to a subject. Accordingly, in one embodiment, the inventionprovides a method of treating Huntington's disease in a subject, byadministering to the subject a composition comprising exosomes loadedwith hydrophobically modified oligonucleotide that reduces expression ofa mutant allele of the huntingtin gene in the subject. In an exemplaryembodiment, the oligonucleotide is an siRNA. In a method of treatingHuntington's disease, it may be desirable to utilize exosomes thattarget neuronal cells. In some embodiments, such exosomes are derivedfrom neuronal cells. In other embodiments, the exosomes are derived fromstem cells, e.g., iPS cells. In other embodiments, the exosomes aresynthetic exosomes. In some embodiments, the exosomes contain atargeting peptide which targets the exosomes to neuronal cells. In otherembodiments, the exosomes do not contain a targeting peptide.

In another example, in a method of treating Amyotrophic LateralSclerosis (ALS), hydrophobically modified oligonucleotides targeting amutant allele of the superoxide dismutase 1 (SOD1) gene are incorporatedinto the exosomes that are administered to a subject. Accordingly, inone embodiment, the invention provides a method of treating ALS in asubject, by administering to the subject a composition comprisingexosomes loaded with hydrophobically modified oligonucleotide thatreduces expression a mutant allele of SOD1 in the subject. In anexemplary embodiment, the oligonucleotide is an siRNA. In a method oftreating ALS, it may be desirable to utilize exosomes that targetneuronal cells. In some embodiments, such exosomes are derived fromneuronal cells. In other embodiments, the exosomes are derived from stemcells, e.g., iPS cells. In other embodiments, the exosomes are syntheticexosomes. In some embodiments, the exosomes contain a targeting peptidewhich targets the exosomes to neuronal cells. In other embodiments, theexosomes do not contain a targeting peptide.

Other non-limiting examples of diseases or disorders that can be treatedby administration of exosomes loaded with hydrophobically modifiedoligonucleotide cargo in accordance with the methods of the inventionare set forth in Table 1. Exemplary genes that can be targeted by thehydrophobically modified oligonucleotide cargo in each instance are alsoprovided.

TABLE 1 Exemplary Disorders Treatable by Administration of ExosomesLoaded with Hydrophobically Modified Oligonucleotide Cargo Disorder GeneTarget(s) Huntington's disease Htt Amyotrophic lateral sclerosis SOD1,C9orf72 Spinocerebellar ataxias (SCA) ATXN1 SCA1 SCA3 ATXN3 SCA6 CACNA1Parkinson's disease a-synuclein LRRK2 GAD67 Alzheimer's disease APP PS1Multiple sclerosis Act1 Prion disease PrP(C) Crohn's disease, IBD ICAM-1Ulcerative colitis ICAM-1 Rheumatoid arthritis TNF-α HIV Gag CMVretinitis CMV mRNA Ovarian cancer C-rat Cancer Hif-1α CML c-Myb Solidcancer: ovarian and others c-Raf Cancer (e.g., malignant Bcl-2 melanoma,NHL, CLL, MM, NSCLC) Cancer Hsp27 Solid cancer Survivin Solid tumorseIF-4E Prostate, breast and lung cancers Clusterin Malignant gliomaTFT-β2 Lymphomas and solid cancers Ribonucleotide reductase R1 Renalcancer Ribonucleotide reductase R2 Solid tumors XIAP Metastatis renalcancer DNA methyltransferase Head and neck cancer DNA methyltransferaseColon cancer, breast cancer and MDM2 brain cancer Prostate cancer IGFBP2and IGFBP5 Prostate cancer HSP27 Asthma IL4R-alpha HypercholesterolemiaApo-B100 Diabetes PTP-1B HCV HCV IRES Type II diabetes Glucagon receptorRestenosis c-Myc mRNA Polycystic kidney disease c-Myc mRNA Cancer c-MycmRNA Cardiovascular disease c-Myc inhibitor Myasthenia gravis AchECancer/metabolism Cyp 3A4 Muscular dystrophy Dystrophin Multiplesclerosis VLA-4 Diabetes type I CD40, CD80, CD86 in dentritic cellsDiabetic retinopathy c-Raf Age-related macular edema VEGF (AMD),Diabetic macular edema Acute kidney injury P53 Pachyonychia congenitalMutant keratin Metastatic melanoma immunoproteasome Liver cancer VEGFKSP Chronic nerve atrophy Caspase 2 Nonarteritic ischemic opticneuropathy Intraocular pressure and glaucoma β-adrenergic receptor 2Ovarian cancer Furin Transthyretin amyloidosis Transthyretin Operablepancreatic ductal Mutated KRAS adenocarcinoma Solid cancers and lymphomaPolo-like kinase Hypercholesterolemia PCSK9 Dermal scarring CTGF HIVinfection CCR5 Familial amyloid polyneuropathy TTR Acromegaly Growthhormone receptor

Exosomes loaded with hydrophobically modified oligonucleotide cargo thatare administered to a subject or used in methods of treatment includethose produced according to any of the methods described herein.

The exosomal compositions described herein can be administered to asubject (i.e., a human or animal subject) by any suitable means. Forexample, appropriate routes of administration include parenteral,intramuscular, intracerebral, intravascular, subcutaneous, ortransdermal. In preferred embodiments, the mode of administration is byinjection, e.g., intramuscular or intravenous injection. A physicianwill be able to determine the mode of administration appropriate for agiven subject.

Exosome administration may be by local or systemic administration. Localadministration, depending on the tissue to be treated, may in someembodiments be achieved by direct administration to a tissue (e.g.,direct injection, such as intratumoral injection, intramyocardialinjection, or injection of neuronal cells or tissue). Localadministration may also be achieved by, for example, lavage of aparticular tissue (e.g., intra-intestinal or peritoneal lavage). Inseveral embodiments, systemic administration is used and may be achievedby, for example, intravenous and/or intra-arterial delivery. In certainembodiments, intracoronary delivery is used.

In some embodiments, subcutaneous or transcutaneous delivery methods areused. Due to the relatively small size, exosomes are particularlyadvantageous for certain types of therapy because they can pass throughblood vessels down to the size of the microvasculature, thereby allowingfor significant penetration into a tissue. In some embodiments, thisallows for delivery of the exosomes directly to central portion of thedamaged or diseased tissue (e.g., to the central portion of a tumor oran area of infarcted cardiac tissue). In addition, in severalembodiments, use of exosomes is particularly advantageous because theexosomes can deliver their payload (e.g., the resident nucleic acidsand/or proteins) across the blood brain barrier, which has historicallypresented an obstacle to many central nervous system therapies. Incertain embodiments, however, exosomes may be delivered to the centralnervous system by injection through the blood brain barrier.

In some embodiments, exosomes are directly infused into the tissue ofinterest. For example, exosomes can be directly infused into the brain,e.g., by intra-striatal injection. As demonstrated herein,intra-striatal infusion of exosomes loaded with hydrophobically modifiedoligonucleotide results in bilateral distribution. The constructs candelivered as a composition, e.g., a pharmaceutical composition, asdescribed herein. The composition may be formulated for parenteral,intramuscular, intracerebral, intravascular (including intravenous),subcutaneous, or transdermal administration. Compositions for parenteraladministration may include sterile aqueous solutions which may alsocontain buffers, diluents and other suitable additives. The constructsof the invention may be formulated in a pharmaceutical composition,which may include pharmaceutically acceptable carriers, thickeners,diluents, buffers, preservatives, and other pharmaceutically acceptablecarriers or excipients and the like in addition to the exosomes.

A “pharmaceutically acceptable carrier” (excipient) is apharmaceutically acceptable solvent, suspending agent or any otherpharmacologically inert vehicle for delivering exosomes to a subject.Typical pharmaceutically acceptable carriers include, but are notlimited to, binding agents (e.g., pregelatinised maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodiumstarch glycolate, etc.); or wetting agents (e.g., sodium laurylsulphate, etc.).

The compositions provided herein may additionally contain other adjunctcomponents conventionally found in pharmaceutical compositions. Thus,for example, the compositions may contain additional compatiblepharmaceutically-active materials or may contain additional materialsuseful in physically formulating various dosage forms of the compositionof present invention, such as dyes, flavouring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions providedherein.

A therapeutically effective amount of exosomal compositions areadministered to subjects. The dose may be determined according tovarious parameters, especially according to the severity of thecondition, age, and weight of the patient to be treated; the route ofadministration; and the required regimen. A physician will be able todetermine the required route of administration and dosage for anyparticular patient. Optimum dosages may vary depending on the relativepotency of individual constructs, and can generally be estimated basedon EC50s found to be effective in vitro and in in vivo animal models.

The dose of exosomes administered, depending on the embodiment, rangesfrom about 1.0×10⁵ to about 1.0×10⁹ exosomes, including about 1.0×10⁵ toabout 1.0×10⁶, about 1.0×10⁶ to about 1.0×10⁷, about 1.0×10⁷ to about5.0×10⁷, about 5.0×10⁷ to about 1.0×10⁸, about 1.0×10⁸ to about 2.0×10⁸,about 2.0×10⁸ to about 3.5×10⁸, about 3.5×10⁸ to about 5.0×10⁸, about5.0×10⁸ to about 7.5×10⁸, about 7.5×10⁸ to about 1.0×10⁹, andoverlapping ranges thereof. In certain embodiments, the exosome dose isadministered on a per kilogram basis, for example, about 1.0×10⁴exosomes/kg to about 1.0×10⁹ exosomes/kg. In additional embodiments,exosomes are delivered in an amount based on the mass of the targettissue, for example about 1.0×10⁴ exosomes/gram of target tissue toabout 1.0×10⁹ exosomes/gram of target tissue. In several embodiments,exosomes are administered based on a ratio of the number of exosomes tothe number of cells in a particular target tissue, for exampleexosome:target cell ratio ranging from about 10⁹:1 to about 1:1,including about 10⁸:1, about 10⁷:1, about 10⁶:1, about 10⁵:1, about10⁴:1, about 10³:1, about 10²:1, about 10:1, and ratios in between theseratios. In additional embodiments, exosomes are administered in anamount about 10-fold to an amount of about 1,000,000-fold greater thanthe number of cells in the target tissue, including about 50-fold, about100-fold, about 500-fold, about 1000-fold, about 10,000-fold, about100,000-fold, about 500,000-fold, about 750,000-fold, and amounts inbetween these amounts. In certain embodiments, the dosage is from 0.01mg/kg to 100 mg per kg of body weight. For example, a daily dose canrange from about 0.1 to 50 mg per kg, preferably from about 0.1 mg/kg to10 mg/kg of body weight, according to the potency of the specificconstruct, the age, weight and condition of the subject to be treated,the severity of the disease and the frequency and route ofadministration. Different dosages of the construct may be administereddepending on whether administration is by intramuscular injection orsystemic (intravenous or subcutaneous) injection. In an exemplaryembodiment, the dose of a single intramuscular injection is in the rangeof about 5 to 20 μg. In another exemplary embodiment, the dose of singleor multiple systemic injections is in the range of 10 to 100 mg/kg ofbody weight.

In several embodiments, the exosomes are delivered in a single, bolusdose. In some embodiments, however, multiple doses of exosomes may bedelivered. In certain embodiments, exosomes can be infused (or otherwisedelivered) at a specified rate over time. Due to construct clearance(and breakdown of any targeted molecule), the patient may have to betreated repeatedly, for example once or more daily, weekly, monthly oryearly. Persons of ordinary skill in the art can easily estimaterepetition rates for dosing based on measured residence times andconcentrations of the construct in bodily fluids or tissues.

VII. Kits

In another aspect, the present invention provides a kit for producing apopulation of exosomes loaded with hydrophobically modifiedoligonucleotide.

In one embodiment, the kit includes a population of exosomes andinstructions for loading the exosomes with hydrophobically modifiedoligonucleotide.

In another embodiment, the kit includes a hydrophobically modifiedoligonucleotide and instructions for incorporating the oligonucleotideinto exosomes.

In another embodiment, the kit includes a composition comprising apopulation of exosomes loaded with hydrophobically modifiedoligonucleotide, and instructions for administration of the compositionto a subject.

In another embodiment, the kit includes a population of exosomes, and ahydrophobic moiety suitable for conjugation to an oligonucleotidemolecule, together with instructions for preparing a hydrophobicallymodified oligonucleotide, and instructions for loading thehydrophobically modified oligonucleotide into exosomes.

The kits of the invention can optionally contain additional componentsuseful for performing the methods of the invention.

By way of example, the kits can contain reagents suitable for incubatingexosomes with hydrophobically modified oligonucleotides. In anotherexample, the kits can contain reagents suitable for purification ofexosomes prior to or subsequent to loading with hydrophobically modifiedoligonucleotides.

In another embodiment, the kits of the invention provide reagents and/orinstructions for determining the loading efficiency of hydrophobicallymodified oligonucleotides into exosomes.

The invention is further illustrated by the following examples, whichshould not be construed as limiting. The entire contents of allreferences, patents and published patent applications cited throughoutthis application are hereby incorporated by reference in their entirety.

EXAMPLES

The experiments described herein demonstrate a simple and robust methodto efficiently load exosomes with oligonucleotide cargo. Loaded exosomesmediated efficient and non-toxic internalization of hydrophobicallymodified siRNA (hsiRNA) and dose-dependent silencing. This data providesa path to utilize exosomes as highly efficient vehicles for delivery ofoligonucleotides in vivo and in vitro.

Example 1: Exosome Production, Purification and Quality Control fromConditioned Media of U87s, BMDCs, HeLa, Huh7 Cells

Exosomes were produced from conditioned media derived by growing severaltypes of cultured cells, including U87, BMDCs, HeLa, and Huh7. Media wasconditioned for various periods of time (2 hours to 72 hours). Exosomesfrom conditioned media were purified using a differentialultracentrifugation method (Thery, C., et al., Isolation andcharacterization of exosomes from cell culture supernatants andbiological fluids. Curr Protoc Cell Biol, 2006. Chapter 3: Unit 3 22).The differential centrifugation protocol is outlined in FIG. 1A.Briefly, cells were cultured in exosome free media. The media washarvested, and centrifuged at 300 g for 10 minutes to remove cells. Themedia was then centrifuged at 2000 g for 10 minutes to remove anyremaining dead cells, and was subsequently centrifuged at 10000 g for 30minutes to remove cellular debris. The supernatant was filtered using a0.2 μm filter, and the filtrate was spun in an ultracentrifuge at100,000 g for 70 minutes to pellet exosomes. The pellet was washed inPBS, and the sample was spun in an ultracentrifuge at 100,000 g for 70minutes. The pellet contained an isolated population of exosomes.Alternatively exosomes were purified using commercially available kitslike ExoQuick (System Bioscience) or Exosome Purification kit (LifeTechnology). In addition, exosomes can be purified by ultrafiltration orgel filtration (Thery, 2006).

Purified exosome size and number was analyzed by light scattering usingNanosight (Malvern). The results of this analysis are presented in FIG.1B. The average size of the exosomes was between 30-120 nm, with most ofthe preps having an average size of ˜100-120 nM. In addition, electronmicroscopy was used to confirm exosome structure and integrity. Arepresentative electron micrograph is shown in FIG. 1C. In summary,exosomes can be produced by wide range of cells and different methodscan be used to purify a relatively homogeneous population, which can beused for loading of the vesicles with hydrophobically modifiedoligonucleotides.

Example 2: Efficient Incorporation of Hydrophobically Modified siRNAsinto Exosomes Purified from U87 Cells

Exosomes have been previously demonstrated to efficiently transfernatural RNAs between cells. It has been shown that exosomes can be usedas delivery vehicles to deliver synthetic RNAs to cells, however, theefficiency of loading synthetic RNA into cells has been very low. Forexample, it has been reported that exosomes can be loaded withnon-natural RNAs by several methods, including (1) making exosomes fromcells over-expressing or pre-transferred with artificial RNAs, (2)electroporation or lipofection of RNAs into purified exosomes, and (3)co-ultra-precipitating exosomes with siRNAs. The efficiency of each ofthese methods is very low. It has been reported that loading exosomeswith oligonucleotide using electroporation results in less than 1-2% ofoligonucleotides being delivered (El-Andaloussi, S., et al.,Exosome-mediated delivery of siRNA in vitro and in vivo. Nat Protoc,2012. 7(12): p. 2112-26) while interfering with exosome integrity(Kooijmans, S. A., et al., Electroporation-induced siRNA precipitationobscures the efficiency of siRNA loading into extracellular vesicles. JControl Release, 2013. 172(1): p. 229-238). Other labs have usedco-ultracentrifugation, resulting in less than one molecule of small RNAloaded per 1000 exosomes, and still demonstrated some functionaloutcomes (Bryniarski, K., et al., Antigen-specific, antibody-coated,exosome-like nanovesicles deliver suppressor T-cell microRNA-150 toeffector T cells to inhibit contact sensitivity. J Allergy Clin Immunol,2013. 132(1): p. 170-81). Hydrophobic modifications of oligonucleotideshave been used to enhance delivery to cells. The experiments describedherein were designed to explore whether hydrophobic modification ofoligonucleotides can be used as way to load exosomes with artificialoligonucleotides such as RNAs.

Exosomes isolated according to Example 1 were loaded withhydrophobically modified siRNA (hsiRNA). The exosome loading andpurification scheme are depicted in FIG. 2A. Briefly, exosomes (100 μl @2.4 μg/μl) were added to a microcentrifuge tube with hsiRNA (10 μM), andwere incubated at room temperature for 30 minutes. Tubes were spun at100,000 g for 70 minutes. The pellet was washed in 1 mL PBS, and thetubes were spun for a second time at 100,000 g for 70 minutes. Thesecond pellet was washed in 1 mL of PBS, and the tubes were spun for athird time at 100,000 g for 70 minutes. The third pellet containedexosomes loaded with hsiRNA.

The sterol conjugates stabilized Cy-labeled—siRNAs (10 uM finalconcentration) were mixed with exosomes derived from U87 cellconditioned media and incubated for 30 minutes at room temperature (RT).5 minutes to 3 hours incubation times at different temperatures (rangingfrom RT-37° C.) were tested and produced similar results. We decided toevaluate whether hsiRNAs described above can be loaded into exosomes.Indeed, when the exosome preparation was incubated with 10 uMDy547-hsiRNA for 1 hour at 37° C., we observed ˜25% of the moleculesassociated with the exosomal pellet after ultracentrifugation. Inaddition, almost no RNA was released in the media after the 3^(rd) spinindicating that binding is stable. This data is depicted in FIG. 2B. Thesize distribution and Z-potential of exosomes was measured before andafter hsiRNA loading (FIG. 3C). A slight shift in size was observed, andan expected change in Z potential (from −5 to −24), oligo fluorescencetracking with the larger exosomes, consistent with exosome associationwith negatively charged oligonucleotides. To separate exosome-associatedRNA from non-bound hydrophobic siRNA the ultracentrifugation wasperformed again. A significant part of the fluorescence appeared inpellet (˜25%). The pellet was bright pink confirming that fluorescentlylabeled siRNAs were associated with the exosomes. Ultracentrifugation ofthe hsiRNA alone did not generate any visible precipitant. The pelletwas re-suspended in PBS and washed twice. After a secondre-centrifugation, the majority of the compound stayed associated withexosomes. The loaded exosomes have slightly shifted Nanosight profileswith a majority of vesicles maintaining the original size. The Zetamembrane potential for loaded exosomes was decreased from −5.7 to −21.2mV, confirming negatively charged RNA association with exosomalmembrane. This data supports efficient and productive loading ofexosomes with chemically synthesized hsiRNA.

To further evaluate whether s majority of hydrophobically labeled siRNAsare associated with the surface of exosomes or internalized, U87produced exosomes were incubated with biotinylated, hydrophobicallymodified siRNAs. The streptavidin gold particles in combination withelectron microscopy were used to detect hydrophobic siRNA localization(FIG. 2D). Gold particles were detected both inside and on the surfaceof exosomes confirming the oligonucleotides are both internalized andare bound to the exosomal membrane.

This data together indicated that hydrophobically modified siRNAefficiently associates with exosomes with more than 25% of addedoligonucleotides being incorporated in vesicles.

Examples of hydrophobic oligonucleotide modifications are depicted inFIG. 3.

Example 3: Fractionation of Exosome-siRNA Complexes by Gel-Filtration

To further evaluate the percentage of hydrophobically modified oligosassociated with exosomes, the oligo-exosome mixtures were fractionatedon a Sephacryl S-1000 gel filtration column (FIG. 4). Exosomes wereeluted in a void volume. Several preps were fractionated using thismethod. Non-Targeting Control sterol conjugated siRNA was incubated withU87 derived exosomes for 1 hour at 37° C. Approximately 43% of oligostayed associated with exosomes after gel filtration. Thesterol-conjugated siRNA targeting huntingtin was incubated with sameexosomal prep and showed ˜26% of oligo associated with exosomes.Consistent loading of hydrophobically modified oligonucleotides withexosomes (>25%) was confirmed with multiple siRNAs and antisense,multiple exosomal preps from different cell types by different methods,indicating that hydrophobic modification of oligonucleotides is a viableapproach for loading artificial RNAs and DNAs into exosomes.

Example 4: Exosome Formulated Hydrophobic siRNAs are EfficientlyInternalized by HeLa Cells, Mimicking Intracellular Localization ofUnloaded Exosomes

The prior examples demonstrate efficient loading of hydrophobicallymodified hsiRNA into exosomes by different methods. This exampleaddresses whether the presence of the hydrophobically modified oligo canimpact exosome trafficking, as hsiRNAs are capable of directinternalization. It was important to demonstrate that this method ofexosome loading did not interfere with exosome biology and did noteffect pathways normally utilized for exosome cellular trafficking.Exosome uptake in HeLa cells was studied (FIG. 5). Exosomes were labeledwith PKH67 lipophilic dye (Sigma) and their kinetics of internalizationwere followed by confocal microscopy. Exosomes were internalized anddetected within the cells in 6-12 hours.

As depicted in FIG. 5, intracellular localization of hsiRNA loadedexosomes closely resembled exosomes alone but was distinctly differentfrom naked hsiRNAs. While hsiRNA was characterized by membrane anddiffuse cytoplasmic vesicular presence, exosome loaded hsiRNAs showeddistinct asymmetric peri-nuclear and no membrane localization. Inaddition, while hsiRNA was internalized instantaneously within minutesof exposure, no detectable internalization with exosome-formulatedhsiRNA was observed for hours (similar to native exosomes), indicatingthat an alternative trafficking pathway was used. Taken together, thisdata demonstrates that hydrophobically modified oligonucleotide-loadedexosomes resemble native exosomes and this method can be used toefficiently load exosomes without interference with their nativetrafficking abilities. This approach can be highly valuable insituations in which exosomes are considered as a potential therapeutic.

Example 5: Exosome Formulated Hydrophobic siRNA Mediates Gene Silencingin HeLa Cells

This example demonstrates that uptake of siRNA loaded exosomes resultsin productive gene silencing. Sterol modified siRNA targeting PPIB, andnon-targeting control, were incubated with U87 derived exosomes.SiRNA-exosome complexes were purified by ultracentrifugation and siRNAloading and exosome integrity was confirmed by Nanosight. HeLa cellswere treated with hydrophobically modified siRNA alone, siRNA-exosomescomplexes, and non-loaded exosomes. Only PPIB-siRNA loaded exosomes showdose dependent silencing of PPIB (Cyclophilin B), as depicted in FIG.6A. The presence of exosomes enhanced efficacy as demonstrated bycomparison with hydrophobically modified siRNA alone (FIG. 6B). Thelevel of PPIB expression was determined by QuantiGene Assay andnormalized to a house keeping gene. Thus exosome mediated siRNA uptakeresults in strong and specific gene silencing and can be used as apotential vesicle for therapeutic applications.

Example 6: Efficient Internalization of Exosome Formulated HydrophobicsiRNAs by Primary Neurons and Exosome-Mediated Silencing of theHuntingtin Gene

Hydrophobic siRNAs targeting huntingtin were loaded in U87 derivedexosomes, purified by ultracentrifugation, and used to treat primarycortical neurons. Similar to the previously described experiment in HeLacells, exosomes promoted uptake of fluorescently labeled siRNAs intoprimary neurons, and this uptake resulted in potent and specific geneknown-down, as shown in FIG. 7. These experiments validate thathydrophobic modification of oligonucleotides is an efficient method forloading of exosomes, and the resulting hsiRNA-exosome complexesdemonstrated efficient cellular uptake and gene specific silencingindependent of the target gene and type of cells used.

Example 7: Exosome Formulated siRNAs Efficiently Distribute Through theBrain and Result in Targeted Delivery to Majority of Neurons

hsiRNA-exosome complexes were further studied to determine whether theyhad the ability to distribute through the animal brain in vivo.Injection of hydrophobically modified siRNA resulted in limited braindistribution (majority of compounds stayed around the site ofadministration), and significant compounds associating with cellularmatrix and some cellular uptake. Interestingly exosome mediated hsiRNAdelivery into brain resulted in uniformly distribution throughout thebrain and preferential delivery to neurons (FIG. 8, stained green withNeuN). This homogenous distribution represents a major breakthrough fortherapeutic oligonucleotide delivery and confirms tissue culture datathat loading of exosomes with hydrophobically modified oligonucleotidedoes not interfere with its ability to promote targeted and productivecellular uptake.

Example 8: Efficient Loading of Exosomes with Hydrophobically ModifiedsiRNAs (hsiRNAs)

In another experiment, exosomes were purified from conditioned medium ofglioblastoma U87 cells by differential centrifugation (FIG. 9A) (Theryet al., 2006). Exosome enrichment was confirmed by size exclusionchromatography of conditioned medium, from which exosomes were purified.(FIG. 9D). Nanoparticle tracking analysis (NTA) showed that the purifiedexosomes had the expected size distribution peaking at 140 nm indiameter. Electron microscopy revealed the cup shape and double membranefeatures characteristic of exosomes (FIGS. 9B and C).

In order to explore if incorporation of hydrophobic modifications intosiRNAs can be used to promote exosome loading, hydrophobically modifiedsiRNAs (hsiRNAs) were used, as described by Byrne et al. Novelhydrophobically modified asymmetric RNAi compounds (sd-rxRNA)demonstrate robust efficacy in the eye. J. Ocul. Pharmacol. Ther. 29,855-64 (2013). The hsiRNAs are asymmetric compounds, with a short duplexregion (15 base-pairs) and a single-stranded fully phosphorothioatedtail. All pyrimidines were modified with 2′-fluoro and 2′-O-methylmodifications and the 3′ end of the passenger strand was conjugated toTeg-Cholesterol (FIG. 10A). The cholesterol enables quick membraneassociation, while the single-stranded phosphorothioated tail isessential for cellular internalization by a mechanism similar to thatused by conventional antisense oligonucleotides (data not shown).

To silence Huntingtin (HTT) expression, hsiRNA HTT10150, targetingposition 10150 of the HTT mRNA sequence, was used. Sequences andchemical modification patterns of compounds used in this study aredescribed in Table 2, below.

TABLE 2  Hydrophobic modification patterns of exemplary siRNA CompoundCompound Conjugate Conjugate Gene Name Type Strand Sequence 5′-3′ 5′ 3′Non NTC hsiRNA s mA.mC.A.A.A.mU.G.A.mU# Cy3 Teg- Targeting mU#mACholesterol Control as PmU.A.A.fU.fC.G.fU.A.fU. fU.GU#mC#A#A#mU#mC#AHuntingtin HTT siRNA s mC.mA.G.mU.A.A.A.G.A.G. Cy3 AmU.mU#mA#mA1 asPmU.fU.A.A.fU.fC.fU.fC. fU.fU.fU.A.fC.fU#G#A#fU# A#fU#A HuntingtinHTT10150 hsiRNA s mC.mA.G.mU.A.A.A.G.A.G. Cye Teg- A.mU.mU#mA#mA1Cholesterol as PmU.fU.A.A.fU.fC.fU.fC. fU.fU.fU.A.fC.fU#G#A#fU# A#fU#A

The loading of exosomes with hsiRNA was performed by the co-incubationof Cy3-HTT10150 (10 μM) with freshly purified U87-derived exosomes for90 min at 37° C. hsiRNAs-loaded exosomes were separated from non-boundhsiRNAs by ultracentrifugation (FIG. 10B). Ultracentrifugation ofexosomes after incubation with CY3-hsiRNA resulted in production of abright red pellet (FIG. 10B) with some of the fluorescence remaining insolution. The ultracentrifugation of hsiRNA in PBS did not produce anypellet indicating that pelleted hsiRNAs are indeed associated withexosomes (FIG. 10C). Approximately 30% of hsiRNA were associated withexosomes based on comparison of fluorescence present in the pellet vssolution (FIG. 10C. To evaluate stability of the hsiRNA-loaded exosomes,the pellet was suspended in PBS followed by a secondultracentrifugation. About 80% of Cy3 fluorescence remained in thepellet confirming a stable interaction between hsiRNA and exosomes(11A). FIG. 11B shows representative pictures of ultracentrifugednon-formulated hsiRNA (left tube) or hsiRNA-loaded exosomes (righttube). In addition, the presence of the hydrophobic modification (e.g.,cholesterol) was essential for the association of siRNA and exosomes, asnon-cholesterol conjugated hsiRNA, of the same chemical composition, wasnot pelleted with exosomes (FIG. 11C). Substitution of cholesterol toother hydrophobic modifications can affect the efficiency of loading(data not shown).

In summary, co-incubation of hsiRNAs and exosomes resulted in efficientloading of hsiRNA into exosomes, and the observed loading was dependenton the presence of the hydrophobic modification, (e.g., cholesterol).

Example 9: Characterization of hsiRNA-Loaded Exosomes

hsiRNA loading was evaluated to see if it interfered with physicalproperties of the exosomes. First, measurement of the fluorescenceintensity in relation to particle numbers (Nanosight), revealed anestimate of about ˜1000-3000 hsiRNA per exosome (FIG. 12A). To estimatethe surface area of exosome occupied by hsiRNA, 130 nm was used as anaverage diameter and ˜53100 nm² as surface area of an exosome. If thediameter of the RNA duplex is 2 nm with approximately a 4 nm² footprint(Watson et al. Molecular Structure of Nucleic Acids. A Structure forDeoxyrribose Nucleic Acid, Nature 171, 737-738 (1953)), then 1000 hsiRNAper exosome will occupy ˜8% of the surface area of an exosome.

Next, the impact of hsiRNA loading on particle size and charge wasevaluated (FIG. 12). Nanoparticle tracking analysis of unloaded andloaded exosomes indicates similar particle size distribution (FIGS. 12 Aand C), and electron microscopy showed similar shapes indicating thathsiRNA association did not interfere with exosome integrity (FIG. 12B).A slight decrease in a Zeta potential of the hsiRNA-loaded exosomes,compared to unloaded exosomes, (from −13 to −30 mV) is consistent withadditional, negatively charged hsiRNA presence on the surface of theexosomes (FIG. 12A). To evaluate localization of hsiRNA on exosomes,exosomes were loaded with a biotinylated HTT10150, labeled withstreptavidin-gold and analyzed by electron microscopy. hsiRNA weredetected both at the surface and in the lumen of exosomes (FIG. 13A-C).Taken together, these data suggest that exosomes loaded with hsiRNA arenot altered in their physical properties and overall integrity.

Example 10: HTT10150-Loaded Exosomes Traffic Efficiently into PrimaryCortical Neurons and Induce Potent HTT mRNA Silencing

hsiRNA labeled with Cy3 (red) and exosomes labeled with a lipophilic dyePKH67 (green), were added to primary cortical neurons and their uptakewas evaluated over time using confocal microscopy (FIG. 14).hsiRNA-loaded exosomes efficiently trafficked into primary neurons withapproximately 62% of hsiRNAs co-localizing with exosomes at 24 hours.

To assess whether exosome mediated hsiRNA delivery would result inproductive gene silencing, mouse primary cortical neurons were treatedfor 7 days in the presence of unloaded, NTC-loaded and HTT10150-loadedexosomes (FIG. 15A). Dose dependent HTT mRNA silencing was observed upontreatment with HTT10150-loaded exosomes but not controls with anestimated IC₅₀ the low nM range. This suggests that exosomes supportefficient neuronal delivery and do not interfere with RISC assembly andgene silencing induced by hsiRNA cargo. The HTT mRNA silencing wasobserved when cells were treated with at least 50×10⁷ exosomes per well.

As exosome internalization is a relatively slow process and there was anon-linear efficiency of internalization (FIG. 15B), the fraction oftherapeutic cargo that was delivered to primary neurons after 4 days wasestimated by tracking the absolute fluorescence associated with thecells (FIG. 15B). There was a higher fraction of exosomes (19% ofexosomes) internalized by the cells at low hsiRNA concentrations than athigh concentration (1.4% of exosomes) (FIG. 15C). This supports thenotion that neurons have a limited capacity to traffic exosomes,consistent with previously observed slow uptake kinetics (FIG. 14). Slowand saturatable trafficking of exosomes in neurons might be an advantagefor in vivo delivery and promote wide distribution of formulatedoligonucleotides through the brain.

As observed by electron microscopy, hsiRNA are detected both in thelumen and at the surface of exosomes. The hsiRNA-loaded exosomes complexseems to be stable during preparation, and the presence of hsiRNA didnot interfere with the physical (size, charge, appearance) andfunctional (trafficking, brain distribution) properties of the exosomes(FIGS. 12 and 14). Simple co-incubation of hsiRNA and exosomes resultsin robust and highly reproducible loading of exosomes, independently ofthe sequence of compounds used. This methodology can be easily appliedto loading of other classes of therapeutic oligonucleotides, includingmiRNA, antisense, antagomirs, or aptamers, as long as oligonucleotidesare stable and hydrophobically modified. The simplicity of the protocolmakes it attractive for the use of exosomes as delivery vehicles foroligonucleotide therapeutics.

Example 11: Bilateral Distribution of Cy3-hsiRNA-Loaded Exosomes UponUnilateral Brain Infusion

To monitor the in vivo bio-distribution of hsiRNA delivered by exosomes,Cy3-hsiRNA-loaded exosomes were infused directly into mouse striatum for7 days (0.5 μg/day). Interestingly, unilateral infusion resulted inbilateral distribution of hsiRNA-loaded exosomes throughout the brain,with Cy3-hsiRNA detectable in striatum and cortex on both sides of thebrain (FIG. 16). The brains of animals infused with non-Cy3-labeledhsiRNA-loaded exosomes were used as a normalization control to insurethat observed fluorescence was indeed due to distribution of Cy3-hsiRNArather than an increase in tissue fluorescence. Implantation of the pumpinduced a similar degree of structural damage at the site of infusion inall treatment groups.

There was a high level of fluorescence visible around the site ofadministration which might be due in part to a high concentration ofexosomes or/and degradation of exosomes followed by local release ofnon-formulated Cy3-hsiRNA. Higher resolution imaging showed that beyondthe immediate injected area there was a uniform distribution of thehsiRNA-loaded exosomes throughout brain tissue (40× magnification, FIG.16). Confocal fluorescent microscopy of tissues co-stained with NeuN(neuronal marker) showed accumulation of compounds in neurons (FIG. 17).

The intra-striatal infusion of hsiRNA-loaded exosomes resulted inbilateral compound distribution (FIGS. 16 and 17). Formulation of hsiRNAinto exosomes is essential for efficacy, as infusion of the same dose ofnon-formulated oligonucleotides did not affect huntingtin mRNAexpression (FIG. 16). These data support the notion that exosomespromote wide tissue distribution and neuronal uptake of their cargo.

Example 12: hsiRNA-Loaded Exosomes Induce Bilateral HTT mRNA Silencingin Vivo in Mouse Brain

It was next evaluated whether observed bilateral distribution wouldresult in HTT mRNA silencing. HTT10150-loaded exosomes withcorresponding controls were unilaterally infused into the striatum ofwild type mice (n=10) by ALZET pump for 7 days. Compared to PBS injectedmice, HTT10150-loaded exosomes showed a dose dependent and statisticallysignificant decrease of HTT mRNA at both concentrations used. Moreimportantly, the silencing was bilateral, consistent with distributionpattern (FIG. 18A). One way ANOVA with Duns' multiple comparisoncorrection showed that the observed silencing was highly significant inall mice treated with HTT10150-loaded exosomes but not in the controlgroups treated with PBS or aCSF, non-formulated HTT10150 (1 μg/day) andunloaded exosomes (FIG. 18B). The fact that unformulated HTT10150 didnot induce HTT silencing, indicates that the hsiRNA10150 loading intoexosomes was essential for both functional neuronal uptake and braindistribution.

The intra-striatal infusion of hsiRNA-loaded exosomes resulted instatistically significant huntingtin silencing (FIG. 18). The ability ofhsiRNA-loaded exosomes to silence HTT mRNA in both treated andnon-treated sides of the brain was indicative of exosomes mediatedefficient spread of compounds through the brain and highlights theadvantage and therapeutic relevance of exosomes as a delivery vehicle inthe CNS.

The Examples above demonstrate that ipsilaterally introduced hsiRNAformulated exosomes induce HTT mRNA silencing on both sides of the brainat exceptionally low concentrations. 3-7 ug of hsiRNA (infused over aweek) was sufficient to induce ˜35% bilateral silencing. The unexpectedand surprising potency of the hsiRNA loaded exosomes shows that thattheir utilization can enable more potent neuronal uptake routes.

Example 13: hsiRNA-Loaded Exosomes Impact on Neuronal Integrity andImmune Response

Nonspecific effects, including immuno-stimulation and cytotoxicity,represent concerns of using exosomes as a therapeutic delivery vehicle.To evaluate the impact of hsiRNA-exosome administration, the level ofmicroglia activation and cell death were monitored. The morphology ofmicroglia cells, which rapidly transform from a resting to an activestate upon activation was assessed by Iba1 staining, a marker ofneuroinflammation whose expression is restricted to microglia andup-regulated upon brain injury (Imai et al., A novel gene iba1 in themajor histocompatibility complex class III region encoding an EF handprotein expressed in a monocytic lineage., Biochem. Biophys. Res.Commun. 224, 855-862 (1996). Intense Iba1-positive microglia arbor anenhanced staining intensity, enlarged cell bodies and ramifiedprocesses. In addition to 7 day point, the microglia response wasevaluated at 6 hours post hsiRNA-loaded exosomes injection (2 μl), assome of the immune response may be transient and might disappear by the7 day time point.

Mice brain sections were analyzed either 6 h after direct striatalinjection or 7 days after pump infusion of PBS, exosomes alone, andHTT10150-exosomes. The changes in histopathology and morphology ofmicroglia staining were comparable between mice injected with PBS, orexposed to exosomes alone or HTT10150-loaded exosomes for 6 hours (FIGS.19 A and C and FIG. 21). Visual evaluation of treated and untreatedbrains did not show any major inflammatory events (FIG. 19B). Althoughmore precise quantification of resting vs activated microgliademonstrated that intracranial injection with PBS induced a slighthigher level of microglia activation in the ipsilateral side of thebrain, but not in the contralateral side. The number of activatedmicroglia was further increased in exosomes and hsiRNA-loaded exosomessamples suggesting a minor neuro-inflammation enhanced by the presenceof injected exosomes (FIG. 19C). Following 7 days infusion, an increaseof activated microglia cells was observed in the ipsilateral side ofbrain injected with PBS, non-loaded exosomes or HTT10150-loadedexosomes. No active microglia were detected in the contralateral side ofthe brain suggesting an inflammatory response most probably due to theimplantation of the pump (FIGS. 19 D, E, and F and FIG. 21).

Finally, in order to assess the cytotoxicity of hsiRNA-exosomesadministration, PBS, exosomes alone and HTT10150-exosomes were infusedinto murine striatum for 7 days. The impact on neuronal integrity wasmonitored by immunohistochemistry for DARPP32 which is expressed in themajority of striatal neurons known as medium spiny neurons. DARPP32staining was performed on brain coronal sections. Qualitative andquantitative analysis of the coronal sections of each brain showed noapparent change in neuronal density on the sides ipsilateral andcontralateral to the injections, (FIG. 20A-C).

The foregoing experiments describe a simple and robust method toefficiently load exosomes with therapeutic oligonucleotides. Inparticular, co-incubation of exosomes with hydrophobically modifiedsiRNAs (hsiRNAs) results in quantitative and efficient association ofoligonucleotides with vesicles. Loaded exosomes mediated efficient andnon-toxic internalization of hsiRNA and dose-dependent HTT mRNAsilencing in primary cortical neurons. Unilateral, intrastriatalinfusion of hsiRNA-loaded exosomes showed a bilateral brain distributionin both striatum and cortex and bilateral silencing of HTT mRNA in thestriatum. Thus, the Examples presented herein provide a path towards theutilization of exosomes as a native, highly efficient vehicle fordelivery of oligonucleotide therapeutics.

Materials and Methods

The Examples described herein were performed with, but not limited to,the materials and methods below.

Cell Culture

U87 glioblastoma cells were maintained in DMEM (Cellgro #10-013CV)supplemented with 100 U/ml penicillin streptomycin (Invitrogen #15140)and 10% fetal bovine serum (Gibco #26140) at 37° C. 5% CO2. Forpurification of exosomes, media from the glioblastoma cultures wastreated with DMEM 4× supplemented with 400 U/ml penicillin streptomycinand 40% FBS and ultracentrifuged in 70 ml polycarbonate bottles (BeckmanCoulter #355622) overnight at 100,000×g, 4° C. using a Type 45 Ti rotor(Beckman Coulter #339160) and filtered in 0.2 μm filter bottle.

Preparation of Primary Cortical Neurons

Primary cortical neurons were isolated from E15.5 mouse embryos of WT(FVBNj) mice. Pregnant females were anesthetized by IP injection ofAvertin (250 mg/kg weight) followed by cervical dislocation. Embryoswere removed and transferred into ice-cold DMEM/F12 medium (Invitrogen#11320). Brains were removed and meninges were carefully detached.Cortices were isolated and transferred into pre-warmed Papain solutionfor 25 minutes at 37° C. and 5% CO2 to dissolve tissue. Papain solutionwas prepared by dissolving Papain (Worthington #54N15251) in 2 mLHibernate E (Brainbits # HE) and 1 mL EBSS (Worthington # LK003188).Separately, DNase (Worthington #54M15168) was re-suspended in 0.5 mLHibernate E. The final Papain solution contained 0.25 mL of re-suspendedDNase mixed with re-suspended Papain. After the 25 minute incubation,Papain solution was removed and 1 ml NbActiv4 (Brainbits # Nb4-500)supplemented with 2.5% FBS was added to the tissue. Cells were thendissociated by trituration with a fire polished, glass Pasteur pipet.Neurons were counted and diluted at 1×106 cells/ml. For live-cellimaging studies, 2×105 cells were plated in glass bottom dishespre-coated with poly-L-lysine (Sigma # P4707). For silencing assays,1×105 neurons per well were plated on poly-L-lysine pre-coated 96-wellplates (BD BIOCOAT #356515). After overnight incubation at 37° C. and 5%CO2 an equal volume of NbActiv4 supplemented with anti-mitotics, 0.484μl/ml of 5′UtP (Sigma # U6625) and 0.2402 μl/ml of 5′FdU (Sigma #F3503), was added to prevent the growth of non-neuronal cells. Half ofthe volume of media was replaced every 48 hours (with new NbActiv4 withanti-mitotics) until the experiments were performed.

Purification and Characterization of Exosomes

Exosomes were purified from U87 glioblastoma cells as described (Tettaet al., Extracellular vesicles as an emerging mechanism of cell-to-cellcommunication., Endocrine 44, 11-9 (2013)) (FIG. 9A). Conditionedculture medium containing exosomes was harvested and centrifuged at300×g for 10 min. The supernatant was then centrifuged at 10,000×g, 4°C. for 30 min and filtered in a 0.2 μm filter bottle. Medium wascentrifuged in 70 ml polycarbonate bottles for at least 70 min at100,000×g, 4° C. using a Type 45 Ti rotor. Pellets were resuspended in 1ml PBS, and ultracentrifuged for 1 h at 100,000×g, 4° C. in a tabletopultracentrifuge using a TLA-110 rotor (Beckman Coulter #366730). Pelletswere resuspended in PBS for further experiments. For each sample, theconcentration and sizes of particles were identified by monitoring therate of Brownian motion using the NanoSight NS300 system (Malvern)calibrated based on manufacturer's protocol. Samples were diluted atroom temperature in 1 ml PBS and monitored in duplicate for 30s withmanual shutter and gain adjustments. The recorded videos were analyzedwith a Nanoparticle Tracking Analysis (NTA) software. The particles'electronegativity was monitored using the Zetasizer Nano NS (Malvern).Samples were diluted in 1 ml H20 and the zeta potential was monitored inthe universal glass cuvette Dip Cell kit (Malvern # ZEN1002).

Loading of Exosomes with Hydrophobic siRNA (hsiRNA) and FluorescentLabeling

Exosomes were loaded with indicated concentration of hsiRNA in PBS byshaking at 500 rpm, 37° C. for 1 h 30 min. Exosomes were washed fromunloaded hsiRNA by ultracentrifugation for 1 h at 100,000 g, 4° C. in atabletop ultracentrifuge using a TLA-110 rotor (FIG. 10). For in vitrouptake experiments, pellets were resuspended in PBS; for in vitro knockdown analysis, pellets were resuspended directly in cell medium.Following the ultracentrifugation, the loading efficiency was estimatedby monitoring the Cy3-fluorescence of the supernatant and the pelletresuspended in PBS. Samples were transferred in Costar UV-transparentflat bottom 96-well plate (Corning #3635) and fluorescence was measuredusing the Infinite® M1000 Pro microplate spectrophotometer (Tecan) withexcitation and emission wavelengths respectively at 547 nm and 570 nm.For monitoring uptake, of hsiRNA, exosomes were fluorescently labeledwith PKH67 dye (Sigma # PKH67GL-1KT). Briefly, 1 μl of PKH67, at a finalconcentration of 10 μM, was added to exosomes, diluted in 100 μl PBS,and incubated for 30 min at 37° C. Free dye was washed from labeledexosomes using the Exosome Spin Column (MW3000) (Life Technologies#4484449) as indicated by the manufacturer's protocol.

mRNA Quantification

mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix #QS0011). Cells were lysed in 250 μL diluted lysis mixture (Affymetrix#13228) supplemented with 0.167 μg/μL proteinase K (Affymetrix # QS0103)for 30 minutes at 55° C. Cell lysates were mixed thoroughly and 40 μl(˜16000 cells) of lysate were added to the capture plate along with 40μl additional diluted lysis mixture without proteinase K. Probe setswere diluted as specified in the Affymetrix protocol. 20 μl of mouse HTTor PPIB probe set (Affymetrix # SB-14150, # SB-10002) was added for afinal volume of 100 μl.

Tissues were lysed in 300 μl of homogenizing buffer (Affymetrix #10642),supplemented with 2 μg/μl proteinase K per 5 mg tissue punch, andhomogenized in 96-well plate format on the QIAGEN TissueLyser II. 40 μlwere added to the capture plate and mixed with 60 μl of either HTT orPPIB diluted probe sets (Affymetrix # SB-14150, # SB-10002) for a finalvolume of 100 μl, as specified by the manufacturer's protocol. Signalwas amplified according to the Affymetrix protocol. Luminescence wasdetected on either the Veritas (Promega) or the Infinite® M1000 Pro(Tecan) microplate luminometer.

Live Cell Staining

For live cell uptake monitoring, cells were plated at a density of 2×10⁵cells per 35 mm glass-bottom dish. Prior to imaging, cell nuclei werestained in cell medium using the NUCBLUE Live READYPROBE as indicated bythe manufacturer (Life Technologies # R37605). Imaging was performed inphenol red free Hibernate E (Brainbits # HE-Pr). Cells were treated with0.5 μM of Cy3-labeled hsiRNA, and live cell imaging was performed overtime.

Brain Sections Immunohistochemistry Staining

In vivo cytotoxicity and microglia activation were monitored byrespectively staining for immunoreactive DARPP32 and Iba1 proteins.Perfused brains were sliced into 40 μm sections in ice cold PBS with theLeica Vibratome 2000T (Leica Biosystems) through the striatumImmunohistochemistry was performed on every 6th section at roomtemperature unless stated. Iba1-reactive cells were stained byincubating sections in blocking solution (5% normal goat serum and 1%bovine serum albumin) with 0.2% TRITON-X-100 and 0.03% hydrogen peroxidein PBS for one hour. Sections were washed with PBS, and incubated withanti-Iba1 (polyclonal rabbit anti-mouse/human/rat; dilution: 1:1,000 inblocking solution) (Wako #019-19741) overnight at 4° C. Sections werewashed with PBS, and incubated in biotinylated secondary antibody (1:200goat anti-rabbit; Vector Laboratories) in PBS for 10 minutes. Afterwashing with PBS, the Vectastain ABC Kit (Vector) was used, followed bya final PBS wash. The reaction was visualized by 3,3′-diaminobenzidine(DAB) with the Metal Enhanced DAB Substrate Kit (Pierce). DARPP32immunohistochemistry consisted of a 3 minute wash in 3% hydrogenperoxide, 20 minute wash in 0.2% TritonX-100, and a 4 hour incubation in1.5% normal goat serum diluted in 1×PBS. Primary DARPP32 antibody(1:10,000 dilution) (Abcam #40801) in 1.5% normal goat serum was added,and sections were incubated at 4° C. overnight. Secondary anti-rabbitantibody, ABC kit, and DAB reagent were used as described above.Following staining, sections were mounted and visualized by lightmicroscopy. Four images were taken at 20× in the striatum of bothinjected and non-injected sides of each section. The number of DARPP32positive neurons and activated microglia, detected by morphology ofstained cells for Iba1, were quantified using ImageJ. Pictures wereacquired with an epifluorescence Leica DM5500-DFC365FX microscope.

Animal Stereotaxic Injections

Animals were deeply anesthetized prior to injection with 1.2% Avertin.For direct injection in the striatum, wild-type (FVBNj) mice receivedmicroinjections by stereotactic placement into the right striata(coordinates (relative to bregma) were 1.0 mm anterior, 2.0 mm lateral,and 3.0 mm ventral). For 7 day infusion, ALZET osmotic pumps (#1007D,delivery rate 0.5 μl/hour over 7 days) were prefilled with 100 μl ofsample following manufacturer's instructions and primed overnight at 37°C. in a water bath. For analysis of immunoreactive DARPP32 and Iba1 micereceived injections or pump infusion (100 μl) of either PBS orartificial cerebrospinal fluid (2 μl per striata, or 100 μl per pump,n=5 mice), 20-30×10⁸ particles/day of exosomes alone (2 μl per striata,or 100 μl per pump, n=5 mice), or HTT10150-exosomes (2 μl per striata or100 μl per pump, n=5 mice), and euthanized 6 hours or 8 days later. Forthe 7 day infusion study, mice were treated with either PBS orartificial cerebrospinal fluid (100 μl per pump, n=10 mice), 20-30×108particles/day of exosomes alone (100 μl per pump, n=10 mice), 1 μg/dayHTT10150 alone (100 μl per pump, n=10 mice) and 1 μg/day of NTC siRNAassociated with 20-30×10⁸ particles/day of exosomes (100 piper pump,n=10 mice) 0.5 or 1 μg/day of HTT10150 associated with 20-30×108particles/day of exosomes (100 μl per pump, n=10 mice/treatment group)and euthanized 8 days later. Brains were harvested and sliced into three300 μm coronal sections. One 2 mm punch was taken from the striatum andcortex from each hemisphere of each section and placed in RNAlater(Ambion # AM7020) for 24 hours at 4° C. Each punch was processed as anindividual sample for the QuantiGene 2.0 Assay analysis. All animalprocedures were approved by the University of Massachusetts MedicalSchool Institutional Animal Care and Use Committee (IACUC, protocolnumber A-2411).

Confocal Imaging

For the analysis of hsiRNA uptake in vitro, mages were acquired with aLeica DM IRE2 confocal microscope using a 63× oil immersion objective.For the study of distribution in brain, images of labeled sections wereacquired with a CSU10B Spinning Disk Confocal System scan head (SolamereTechnology Group) mounted on a TE-200E2 inverted microscope (Nikon) witha 60× Plan/APO oil lens and a Coolsnap HQ2 camera (Roper). Images wereprocessed using ImageJ (1.47v) software and the percentage ofcolocalization was calculated based on Manders Overlap Coefficient usingthe Manders Coefficients plugin in ImageJ.

Size Exclusion Chromatography

Exosomes from U87 were purified as previously described. 50 μl of cellconditioned medium or 50 μl of exosomes were injected onto BioSepSEC-s4000 column (size exclusion column, pore size 500 Å) on Agilent1100 HPLC system. Chromatography was conducted with 0.75 ml/min flowrate with PBS as mobile phase. Eluted fractions were monitored at 220 nmwith Agilent DAD G1315B absorbance detector.

Anion Exchange Chromatography

Primary cortical neurons, plated at 1×10⁵ cells/well in 96-well plate,were treated with Cy3-labeled hsiRNA with or without exosomeformulation. After 4 days of incubation, both medium, transferred into anew 96 well plate, and cells were lysed in EpiCentre Cell and TissueLysis Solution in the presence of proteinase K. SDS from the lysisbuffer was precipitated by 3 M KCl and pelleted at 5000 g. Supernatantwas injected onto anion exchange Dionex DNAPac P100 column on Agilent1100 HPLC system. Chromatography was conducted at 1 ml/min flow rate in50% water, 50% acetonitrile, 25 mM Tris (pH=8.8), 1 mM EDTA, saltgradient 10-100% 800 mM NaclO4. Eluted fractions were detected by Cy3fluorescence with Agilent FLD1260 G1321B detector.

Electron Microscopy

The samples and grids for electron microscopy were prepared at roomtemperature unless specified in the method. An equal volume of 4%paraformaldehyde was added to the exosome sample and incubated for 2 h.3 μl aliquots of exosomes were dropped onto grids and incubated in 2%paraformaldehyde for 20 min. The grids were transferred to a wax stripand washed with 100 μl PBS. The grids were incubated in 50 mMglycine/PBS for 5 min and blocked in 5% BSA/PBS for 10 mM in thepresence or absence of 0.1% saponin. The grids were washed in 2×PBS andincubated with 6 or 10 nm streptavidin immune-gold particles diluted1:10-1:20 in 0.5% BSA/PBS for 1 h in the presence or absence of 0.1%saponin. The grids were washed with 3×PBS and incubated in 1%glutaraldehyde for 5 mM Following 8 washes of 2 mM each with H20, thegrids were incubated for 5 min in uranyl oxalate and in 1% methylcellulose:4% uranyl acetate (9:1) for 10 min on ice. Excess liquid wasremoved with a filter paper and the grids were air dried for 5 to 10 mMExosomes were examined in a JEOL 1100 transmission electron microscopeat 60 kV and. Images were obtained with ATM digital camera.

Statistical Analysis

Data analyses were done using GraphPad Prism 6 version 6.04 software(GraphPad Software Inc.). For determination of IC50s, a curve was fittedusing log(inhibitor) vs. response—variable slope (four parameters). Thebottom of the curve was set to be no less than zero and the top of thecurve was set to be no greater than 100. For each mouse experiment, thelevel of knockdown at each dose was normalized to the mean of thecontrol group, which was the non-injected side in those mice treatedwith PBS or artificial CSF. In vivo data were analyzed using theKruskal-Wallis test (one-way ANOVA) with Bonferroni corrections formultiple comparisons. Differences in all comparisons were consideredsignificant at P<0.05.

INCORPORATION BY REFERENCE

The contents of all cited references (including literature references,patents, patent applications, databases and websites) that maybe citedthroughout this application are hereby expressly incorporated byreference in their entirety for any purpose, as are the references citedtherein. The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of immunology, molecularbiology and cell biology, which are well known in the art.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are therefore intended to be embracedherein.

The invention claimed is:
 1. A composition comprising a plurality ofexosomes loaded with an oligonucleotide comprising one or morehydrophobic modifications, wherein the one or more hydrophobicmodifications comprise a hydrophobic moiety conjugated to theoligonucleotide, wherein the hydrophobic moiety is a sterol, GM1, alipid, a vitamin, or a peptide, or a combination thereof, and whereinthe exosomes contain an average of about 1000-3000 oligonucleotides perexosome.
 2. The composition of claim 1, wherein at least 90% of theexosomes are loaded with the oligonucleotide.
 3. The composition ofclaim 1, wherein the oligonucleotide is a synthetic oligonucleotide. 4.The composition of claim 3, wherein the oligonucleotide is siRNA,siRNA-GalNAc, antisense RNA, LNA, hairpin siRNA, PMO, miRNA, miRNAinhibitors, or combinations thereof.
 5. The composition of claim 4,wherein the oligonucleotide is siRNA or miRNA.
 6. The composition ofclaim 1, wherein conjugation to the hydrophobic moiety increases thehydrophobicity of the oligonucleotide by at least 2 orders of magnituderelative to unmodified oligonucleotide.
 7. The composition of claim 1,wherein the oligonucleotide further comprises hydrophobically modifiednucleotides, and wherein at least 30% of the nucleotides in theoligonucleotide are hydrophobically modified.
 8. The composition ofclaim 1, wherein the oligonucleotide further comprises a hydrophobicmodification which is a backbone modification, a ribose modification, abase modification, or a combination thereof.
 9. The composition of claim8, wherein the backbone modification is selected from the groupconsisting of phosphorothioate modifications, phosphorodithioatemodifications, p-ethoxy modifications, methylphosphonate modifications,methylphosphorothioate modifications, alkyl- and aryl-phosphatemodifications, alkylphosphotriester modifications, peptide nucleic acid(PNA) modifications, and locked nucleic acid (LNA) modifications. 10.The composition of claim 8, wherein the ribose modification is2′O-Methyl, 2′Methoxy-ethyl, 2′Fluoro, or 2′FANA.
 11. The composition ofclaim 8, wherein the base modification is phenyl, naphthyl, or isobutyl.12. The composition of claim 1, wherein the oligonucleotide isconjugated to cholesterol, GM1, myristic acid or a derivative thereof.13. The composition of claim 1, wherein the exosomes are derived fromcultured cells.
 14. The composition of claim 13, wherein the exosomesare derived from dendritic cells (DC), B cells, T cells, mast cells,epithelial cells, stem cells, neuronal cells, and tumor cells.
 15. Thecomposition of claim 1, wherein the exosomes are synthetic exosomes. 16.The composition of claim 1, wherein the exosomes comprise a targetingpeptide.
 17. The composition of claim 16, wherein the targeting peptidetargets the exosomes to neuronal cells.
 18. The composition of claim 1,wherein the oligonucleotide comprises a fluorescent label, and whereinthe average number of oligonucleotides per exosome is determined by amethod comprising: (a) purifying the plurality of exosomes loaded witholigonucleotide; (b) measuring fluorescence intensity and exosome numberin the plurality of exosomes purified in part (a); (c) using thefluorescence intensity and the exosome number measured in part (b) todetermine an average number of oligonucleotides per exosome.